WO2020051370A1 - Discovery of novel potent inhibitors of the map3k mekk2 - Google Patents

Abstract

The identification and confirmation of 3 HTS hits with in vitro MEKK2 inhibitory activity using the MEKK2 ATPase HTS assay is shown. Preliminary SAR studies identified additional compounds for inhibiting MEKK2. Use of these compounds for inhibiting MEKK2 is described.

Description

DISCOVERY OF NOVEL POTENT INHIBITORS OF THE MAP3K MEKK2 This invention was made with government support under grant no. U54CA156735 funded by the National Cancer Institute / National Institutes of Health. The U.S. government has certain rights in the invention. BACKGROUND OF THE INVENTION The ERK5 signaling pathway is one of the mitogen-activated protein kinases (MAPK) signaling pathways that translate extra-cellular stimuli into gene expression changes in the cell (1, 2). This pathway is activated in response to oxidative stress, hyperosmolarity, and growth factors, including epidermal growth factor (EGF) (3). ERK5 is activated through phosphorylation by the MAPK kinase MEK5 (MAP2K5). MEK5 in turn is activated through phosphorylation by the MAPK kinase kinases MEKK2 (MAP3K2) and MEKK3 (MAP3K3). How MEKK2 is activated by external stimuli is still not fully understood, but MEKK2 autophosphorylates in response to stimuli and this autophosphorylation is required for activation of MEKK2 (4). MEKK2 also activates the JNK MAPK pathway via phosphorylation of the MAP2K MEK7 which activates JNK (5, 6). Recently, MEKK2 was shown to be methylated at lysine 260 by the methyltransferase SMYD3 (7). However, this methylation did not affect MEKK2 kinase activity and the consequences for the ERK5 and JNK pathways are not currently known. Mouse gene knock-out studies have provided clues to the biological function of the MEKK2/3- MEK5-ERK5 pathway. ERK5 knock-out in mice resulted in embryonic lethality due to defects in cardiac development and angiogenesis (8). ERK5 appears to protect many cell types from stress-induced apoptosis (3, 9, 10). MEK5 gene knock out in mice resulted in lethality apparently due to abnormal cardiac development, similar to the ERK5 knockout mouse (11). MEKK2 knock-out mice displayed normal development and fertility (5, 12). In contrast, disruption of the MEKK3 gene in mice results in embryonic lethality due to cardiac development defects (13). Thus, MEKK2 and MEKK3 integrate different stimuli. MEKK2 has been reported to be activated by cell attachment to fibronectin and to subsequently localize to focal adhesions. MEKK2 knockdown in breast cancer cell lines was shown to stabilize focal adhesions and inhibit cell migration in vitro (14, 15). The role of the MEKK2 in cancer has only relatively recently been explored. In one study linking MEKK2 to cancer, MEKK2 was expressed at 4.4-fold higher level in prostate cancer tissue versus benign tissue (16). Even higher levels of MEKK2 were observed in prostate cancer cell lines. The microRNA miR-520b suppresses tumor formation in breast cancer and hepatocellular carcinoma cells by targeting MEKK2 and Cyclin D1 (17). Knock-down of only MEKK2 expression was able to inhibit the growth of hepatocarcinoma cells in vitro and in vivo. The methylation of MEKK2 by SMYD3 was shown to increase MAP kinase signaling and promote the formation of Ras-dependent carcinomas (7). Restoration of the expression of the tumor suppressor miR17/20a was shown to enhance tumor cell sensitivity to natural killer cell activity through suppressing MEKK2 (18). A survey of primary colorectal cancer (CRC) lesions for MEKK2 expression level by western blotting indicated that MEKK2 was highly expressed in CRC compared to normal mucosa (19). An in vivo mouse xenograft model of breast cancer was used to assess the role of MEKK2 in tumor growth and metastasis (20). Using the breast cancer cell line MDA-MB-231, it was observed that shRNA-mediated knockdown of MEKK2 inhibited activation of ERK5 in response to epidermal growth factor (EGF). Knockdown of MEKK2 expression had no observable impact on the growth of the cells in culture, but strongly inhibited both tumor growth and metastasis in the animal model. The tumors formed by the MEKK2 knock-down cells had increased apoptosis compared to size-matched control tumors. MEKK2 shRNA knockdown in the BT474 breast cancer cell line also resulted in reduced tumor growth in vivo. These data indicated that MEKK2 is required for EGFR- and Her2/Neu-dependent ERK5 activation, tumor growth and metastasis of MDA-MB-231 cells and tumor growth of BT474 cells. Since ERK5 is activated by MEKK2 (via activation of MEK5), it was determined whether shRNA knockdown of ERK5 in MDA-MB-231 cells would show similar inhibition of tumor growth and metastasis as the MEKK2 knockdown. The knockdown of ERK5 in these cells inhibited their ability to metastasize without significantly impacting tumor growth. Thus, MEKK2- mediated activation of ERK5 appeared to regulate metastasis, while another MEKK2- dependent pathway, possibly the JNK pathway, was important for tumor growth. Overexpression of ERK5 protein was found in 20% of 84 human early stage breast cancer tissue samples (21). Overexpression of ERK5 was also correlated to decreased disease-free survival times. Similarly, ERK5 expression was significantly increased in high-grade prostate cancer when compared to benign prostatic hyperplasia (16, 22). In addition, ERK5 expression in tissue taken before and after hormone relapse suggested a correlation between ERK5 activation and hormone-insensitive prostate cancer (22). Forced overexpression of ERK5 in PC3 cells produced cells that were more efficient at forming tumors in mice. In another report, expression of a dominant negative form of ERK5 inhibited the proliferation of myeloma cells and enhanced sensitivity to apoptosis-inducing drugs (23). The microRNA miR-143 appears to be a suppressor of ERK5 protein expression and has been shown to be a tumor suppressor using a mouse model of prostate cancer (24). ERK5, but not ERK1, pathway activation correlated with lymph node metastasis in oral squamous cell carcinoma (25). The discovery of a potent and selective small molecule ERK5 inhibitor has been reported that inhibited tumor growth in mouse models of cancer (26). Inhibiting ERK5 function by an ERK5 inhibitor or by RNAi-mediated knock-down suppressed neuroblastoma cell proliferation in vitro and enhanced the antitumor activity of an ALK inhibitor in both cells and xenograft models (27). Using this ERK5 inhibitor, ERK5 inhibition has also been reported to synergize with HSP90 inhibitors for TNBC cell lines (28). MEK5 may also play a role in tumor development. Elevated tissue expression of MEK5 correlated with bone metastasis and poor prognosis in cases of prostate cancer and benign prostatic hypertrophy (29). Two relatively selective MEK5 inhibitors have been reported, but no in vivo efficacy data were shown (30). Taken together, literature data supports MEKK2 as a novel drug target for certain cancers. Targeting MEKK2 may be advantageous over inhibiting single MAPK pathways since an MEKK2 inhibitor may blunt activation of both the ERK5 and JNK pathways leading to enhanced anti-tumor efficacy. However, no specific and potent small molecule inhibitors of MEKK2 have been reported to date. SUMMARY OF THE INVENTION An aspect of the invention is directed to identification of MEKK2 inhibitors. Another aspect of the invention is directed to a method for identifying MEKK2 inhibitors. Yet another aspect of the invention is directed to a method for screening MEKK2 inhibitors. Still another aspect of the invention is preparation and identification of MEKK2 inhibitors. In another aspect of the invention are compounds that are MEKK2 inhibitors. Yet another aspect of the invention is use of a compound to inhibit MEKK2. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Selectivity of compound 2 in a panel of 50 kinase activity assays. Compound 2 was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 2. Selectivity of compound 1 in a panel of 50 kinase activity assays. Compound 1 was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 3. Selectivity of compound 1e in a panel of 50 kinase activity assays. Compound 1e was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 4. Selectivity of compound 1f in a panel of 50 kinase activity assays. Compound 1f was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 5. Selectivity of compound 1s in a panel of 50 kinase activity assays. Compound 1s was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 6. Selectivity of compound 5a in a panel of 50 kinase activity assays. Compound 5a was tested at 1 mM in duplicate in each assay and the average percent inhibition was plotted for the indicated kinase. Results were sorted by activity with the highest percent inhibition on the left-side of the graphs. Dotted line indicates 50% inhibition. Figure 7. IC₅₀ determinations for compound 1s in selected kinase activity assays. Assays were performed externally using a different source of enzyme in ELISA-based activity assays where the [ATP] was at or near the respective Km values. Calculated IC₅₀ values were 60 nM for MEKK2, 53 nM for MEKK3, >1 mM for MAP2K5 and >10 mM for TAK1-TAB1 and MAP3K5. DETAILED DESCRIPTION OF INVENTION It is to be understood that the methods and compounds described are not limited to the particular methodology, protocols, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compound described herein. For purposes of the description and claims, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers such as those expressing values, amounts, percentages, ranges, subranges and fractions may be read as if prefaced by the word“about”, even if the term does not expressly appear.

Accordingly, unless indicated to the contrary, the numerical parameters set forth are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where a closed or open-ended numerical range is described herein, all numbers, values, amounts, percentages, subranges and fractions within or encompassed by the numerical range are to be considered as being specifically included in and belonging to the original disclosure of this application as if these numbers, values, amounts, percentages, subranges and fractions had been explicitly written out in their entirety. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms, such as "include",

"includes," and "included," is not limiting. As used herein, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. As used herein,“including”,“containing” and like terms are understood in the context of this application to be synonymous with“comprising” and are therefore open-ended and do not exclude the presence of additional undescribed and/or unrecited elements, materials, ingredients and/or method steps. As used herein,“consisting of’ is understood in the context of this application to exclude the presence of any unspecified element, ingredient and/or method step. As used herein,“consisting essentially of” is understood in the context of this application to include the specified elements, materials, ingredients and/or method steps“and those that do not materially affect the basic and novel characteristic(s)” of what is being described. All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the methodologies that are described in the publications, which might be used in connection with the methods, uses and compounds described herein. In the present invention,“inhibitor” refers to a substance having an inhibitory activity. The term“inhibit” when used in reference to activity refers to a partial or complete reduction in activity of a molecule, enzyme, target or the like.“Inhibitor” may also refer to a compound or compounds that interferes with: (1) the expression, modification, regulation or activation of a molecule, enzyme, target or the like, and/or (2) one or more normal functions of a molecule, target or the like. Abbreviations used include: DMSO (dimethyl sulfoxide); MAPK (mitogen-activated protein kinase); MS (mass spectrometry); ATP (adenosine triphosphate); ADP (adenosine diphosphate); SAR (structure-activity relationship); SD (standard deviation); TR-FRET (time-resolved fluorescence resonance energy transfer); and HTS (high throughput screening). The development and validation of an intrinsic ATPase activity assay for MEKK2 and demonstration of its utility as a high throughput assay for the discovery of small molecule inhibitors of MEKK2 is described in Ahmad S, Hughes MA, Johnson GL, Scott JE. Development and validation of a high-throughput intrinsic ATPase activity assay for the discovery of MEKK2 inhibitors. J Biomol Screen. 2013;18:388-99 (31) as stated below. MEKK2 Intrinsic ATPase Activity Assay All compound stock solutions were made in 100% DMSO. Serial dilutions of compounds for IC 50 determinations were initially performed in 100% DMSO in 96-well polypropylene plates, followed by appropriate dilutions in assay buffer (50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM sodium

orthovanadate, 0.5 mM b-glycerophosphate, 2.5 mM DTT, and 0.01% Triton X-100), producing a constant 2.5% DMSO in all wells. Subsequently, 10 mL of the diluted compound (or just 2.5% DMSO in assay buffer for controls) was added to the wells of a 96-well plate followed by 10 mL of MEKK2 enzyme diluted in assay buffer. After a 10-min incubation, the reactions were initiated by addition of 5 mL of ATP (150 mM ultrapure ATP from Promega kit) diluted in assay buffer, and the assay plates were centrifuged at 1000 rpm for 1 min in a Beckman Coulter Allegra X-12R centrifuge. Final concentrations in the assembled standard assay were as follows: 1x assay buffer, 1% DMSO, 30 mM ATP, and 10 nM MEKK2 enzyme. The enzyme reactions were allowed to proceed protected from light for 75 min at 23°C and terminated by the addition of 25 mL of solution 1 (ADP-Glo Reagent, Promega). At this point, assay plates were centrifuged again as described above and incubated at 23°C for an hour. A 50 mL volume of solution 2 (detection reagent) of the Promega kit was added to each well, and the plates were incubated for 1 h at 23°C, protected from light. The plates were read using an endpoint luminescence protocol (measurement interval time: 1.0 s, optic module: LUM plus, gain: 3600, focal height: 13.7 mm, positional delay: 0.1 s) in a BMG. PHERAstar (BMG Labtech, Cary, NC) plate reader. Luminescence data, expressed in relative luminescence units (RLU), were normalized to DMSO (100% activity) and“no enzyme” (0% activity) controls as maximum and minimum responses, respectively. For all MEKK2 assay formats, compound concentration response curves were generated using data points that represent the average of three determinations per concentration, except for the

transphosphorylation assay, in which duplicate determinations were performed. All IC 50 values provided in this report are averages of at least three independent determinations. The IC 50 values and Hill slopes were calculated from concentration-response data using

GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) employing either a four- parameter or a three-parameter (fixed bottom) curve fit. This assay was used to screen a collection of approximately 15,000 commercially available compounds. Three confirmed hits from this screen were identified. Hit compounds 1, 2 and 3 (shown in Table 1) generated IC₅₀ values in the MEKK2 ATPase activity assay of 322 ± 161, 27,500 ± 10,900, and 7,710 ± 1,610 nM (Table 1). All three commercially-available compounds were confirmed for purity and mass by LC-MS. Compound 1 was further confirmed as a hit by re-synthesis. Two completely different MEKK2 assay formats were used to confirm the activity observed in the ATPase assay. The MEKK2 TR-FRET binding assay measured binding to MEKK2 via competitive displacement of a labeled ATP-site binding small molecule. An additional MEKK2 activity assay was employed that detects the native transphosphorylation activity of MEKK2 where kinase-inactive MKK6 was used as a substrate and MEKK2 catalyzed phosphorylation of MKK6 detected by western-style quantitative slot blot and phospho-antibody detection of phosphorylated MKK6 (31). Table 1. Activities of screening hits in different MEKK2 assay formats

Compound 1 produced average potencies of 355 ± 134 nM in the binding assay and 261 ± 14 nM in the transphosphorylation assay format (Table 1). Thus, compound 1 confirmed activity in all three MEKK2 assay formats. Compound 2 interfered with the binding assay and therefore could not be accurately tested in this format. Surprisingly, compound 2 produced an IC₅₀ of 227 ± 102 nM in the transphosphorylation assay, shifting 121-fold more potent compared to the primary ATPase assay. Addition of compound 2 to the MEKK2 enzyme reaction after stopping the reaction did not inhibit the signal indicating that its activity in the transphosphorylation assay was not due to assay method interference. Likewise, no significant assay interference could be detected by the compound itself in the homogeneous ATPase assay. This shift could be due to solubility problems in the ATPase assay or a preferential binding of compound 2 to the enzyme-substrate (MEKK2-MKK6) complex. During kinome profiling experiments by Carna Biosciences, 1 mM compound 2 produced an average of 31% inhibition of an MEKK2/MAP2K7/MAP2K4/JNK2 cascade reaction performed at a high 1 mM ATP while producing <10% inhibition in parallel MAP2K7/JNK2 and MAP2K4/JNK2 assays at the same ATP concentration (data not shown). Thus, compound 2 appears to be a genuine inhibitor of MEKK2. Compound 3 generated similar data in one of the other assay formats with an IC₅₀ of 7,926 ± 3,598 nM in the binding assay. Thus, all three hits were confirmed as MEKK2 inhibitors. A SAR (structure-activity relationship) of compound 1 was carried out to enhance its potency and selectivity. The efforts focused on substitution around the phenol ring and modifying the methoxy group on the chromene ring and assessing those changes on MEKK2 inhibitory activity (Table 2). Table 2. Compound 1 derivatives and MEKK2 inhibitory activities

Replacing the hydroxy group of the phenol ring with a methoxy group (Table 2, compound, 1a) resulted in the complete loss in inhibitory activity, indicating that the hydroxy group is absolutely required for activity. This data may be analogous to a recently reported LRRK2 inhibitor in which modeling and SAR suggested that a critical 4-hydroxy on a phenol group may act as donor and acceptor with conserved residues (33). The R’₂ position of the phenol ring, was substituted with a chlorine, methyl, hydroxy and a fluorine group (Table 2, compounds 1b-e). The presence of a methyl or hydroxy group had little impact on the potency. In contrast, the chlorine or fluorine group resulted in enhanced potencies with IC₅₀s of 60 and 115 nM. The R’₁ position on the phenol group, was substituted with CF₃, methyl and fluorine groups (compounds 1f-h) which resulted in dramatic enhancement of MEKK2 potency with IC₅₀ values of 38, 28 and 39 nM, respectively. Since a fluorine group at the R’1 and R’2 positions was shown to enhance the potency, it was sought to determine if difluorinated analogs of compound 1 would further improve the potency. Difluorinated compounds 1i-j (Table 2,) with fluorines at the R’₁/R’₂ and R’₂/R’₄ positions generated IC₅₀ values of 36 and 27 nM. These compounds were similar in potency as monofluorinated compound 1h. However, substitution of fluorines at the R’₁ and R’₅ positions of the phenolic ring generated compound 1k with an IC₅₀ of 8 nM. Taken together, the enhancement in potency of the mono and difluorinated compounds may be due to the electron withdrawing ability of this group to modulate the hydrogen bonding donor/acceptor of the phenol hydroxyl group. Limited SAR was also explored at the methoxy position of the chromene ring (Table 2). Replacement of the methoxy group with an ethoxy, propoxy, isopropoxy, fluorine, bromine or hydroxy group resulted in compounds 1l-q which gave IC₅₀s of 130, 371, 502, 2102, 151 and 766 nM respectfully. Thus, the ethoxy group at this position resulted in >2-fold improvement in potency which was best among the tested substitutions at this position. Since a methyl or CF₃ group at the R’₁ position on the phenol group dramatically enhanced potency of the hit compound 1, analogs of these compounds containing an ethoxy group on the chromene ring were synthesized. The methyl and CF3 substituted compounds 1r-s generated IC₅₀s that were reduced to 18 nM and 16 nM (Table 2), respectively. It was sought to scaffold jump to explore the activities of related structures by attempting to switch from the iminochromene core to a quinoline core scaffold (Table 3). Table 3. Quinoline analogs of compound 1

The quinoline analog of the original hit compound 1 was synthesized with a secondary amine linking the quinoline to the phenol group (compound 4), but this resulted in an IC₅₀ of 5.6 mM. Switching the quinoline-phenol linking atom to oxygen (compound 5) improved the potency resulting in a MEKK2 inhibitory potency of 1.3 mM. It was sought to improve potency of this quinoline scaffold further by applying knowledge from the SAR of the iminochromene scaffold (Table 2). Thus, we substituted a R’₁ methyl on the phenolic group and ethoxy on the quinoline ring 5 resulting in compound 5a. This compound generated an IC₅₀ value of 84 nM, thus holding promise as a parallel or alternative scaffold to the iminochromene core. With these modifications, this quinoline core analog became only approximately 5-fold less potent than compound 1s, one of the most potent iminochromene analogs. These substitutions on the quinoline core resulted in a 15-fold enhancement of potency which is similar in magnitude to their effect on the iminocoumarin core scaffold in which they enhanced potency 20-fold. Thus, knowledge from the iminochromene SAR may be translated to this new core scaffold. In order to assess the selectivity of these scaffolds as kinase inhibitors, the inhibitory activity of selected compounds was profiled against a panel of diverse kinase activity assays performed by Carna Biosciences. Selected compounds were tested in duplicate at 1 mM compound against a panel of 50 diverse serine/threonine and tyrosine kinase activity assays. The initial hit compound 2 was profiled and inhibited ³50% of the activity (“hit”) in only 8% of the kinase panel (Fig. 1). In contrast, compound 1 inhibited ³50% of the activity in 40% of the kinase panel (20 of the 50 kinase assays), thus indicating low selectivity for this hit (Fig. 2). The R'₂ fluoro analog of compound 1, compound 1e, inhibited ³50% of the activity in 42% of the kinase panel (Fig. 3). Of note, >95% inhibition was obtained in 7 kinase assays whereas the original hit compound produced >95% inhibition in only 3 kinases. These data suggested that the 2’ F substitution resulted in reduced selectivity. In contrast, profiling of compound 1f with the 1’ CF₃ substitution on the phenol group resulted in ³50% inhibition of 8% of the kinases tested (Fig. 4). Thus, the 1’ CF₃ group appeared to impart enhanced selectivity while enhancing activity against MEKK2 by over 8-fold. These data imply a sub- site in MEKK2 that could be exploited to enhance inhibitor selectivity. Profiling of the dual modified analog with a 1’ methyl group on the phenol and ethoxy substitution on the iminocoumarin ring (compound 1s) resulted in hitting 16% of the kinases in the panel (Fig. 5), but it is also twice as potent against MEKK2 compared to compound 1f. Overall, the activity and selectivity profile appeared similar between compounds 1f and 1s. Compound 5a, the quinoline analog of compound 1s, was profiled to determine the effect that the different core scaffold had on selectivity (Fig. 6). This quinoline compound only hit 6% of the kinase panel which were the three kinases JAK2, p70S6K and EphA2. Remarkably, these 3 kinases were the only ones in the panel that were inhibited by ³25%. Thus, this data suggested that the switch to the quinoline scaffold dramatically enhanced selectivity, while still being 3.8-fold more potent against MEKK2 compared to the original hit. Validation of the MEKK2 inhibitory activity of compound 1s using a different source of MEKK2 and a different assay format while also further assessing selectivity of other kinases in the MAPK family (Fig. 7). An MEKK2 transphosphorylation assay was performed by Carna Biosciences that uses MAP2K7 as a substrate and an ELISA for detection of phosphorylated product. In this assay, compound 1s generated an IC₅₀ of 60 nM against MEKK2 which is similar (<4-fold) to the IC₅₀ value determined by the ATPase assay format. The discrepancy in potencies could be due to the use of only the catalytic domain of MEKK2 in the CarnaBio assay, as opposed to the full-length MEKK2 in our assays. This compound also produced an IC₅₀ of 53 nM against the closely related MEKK3, making it a dual inhibitor of MEKK2/MEKK3. This was not unexpected since the catalytic domains of MEKK2 and MEKK3 are 94% homologous (34). Activity against MAP2K5 was also tested and 50% inhibition was not achieved, possibly due to solubility problems in this assay. Based on the dose response curve, we estimated an IC₅₀ of >1 mM against MAP2K5. No activity (>10 mM IC₅₀) was observed against MAP3K5 (ASK1) and TAK1-TAB1 (MAP3K7). The identification and confirmation of 3 HTS hits with in vitro MEKK2 inhibitory activity using the MEKK2 ATPase HTS assay (31) is shown. Preliminary SAR studies for one of the hits resulted in >10-fold more potent analogs with the most potent one having an IC₅₀ of 8 nM for MEKK2. This small set of analogs also led to the identification of analogs with dramatically improved selectivity in a 50 member kinase profiling panel. These SAR studies suggested that substitutions around the phenoxy ring can impart improved selectivity for MEKK2, as well as potency. Scaffolds switched to a quinoline core that not only improved potency over the original hit, but perhaps more importantly dramatically improved selectivity. Typically, a compound for use in inhibiting MEKK2 activity is administered in an amount effective to inhibit MEKK2 activity. The compound or composition including, comprising, consisting essentially or consisting of the compound is administered by any suitable route in a form adapted to such a route, and in a dose effective for the effect intended. The term “therapeutically-effective amount” or“dose” as used herein refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated. Compounds useful in accordance with the invention may be provided as salts with

pharmaceutically compatible counterions (i.e., pharmaceutically acceptable salts). A “pharmaceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound or a prodrug of a compound useful in accordance with this invention. The inhibitors used according to the invention may be administered to a subject alone, or in a mixture with at least one pharmaceutically acceptable excipient which may be an excipient known to those skilled in the art. The pharmaceutically acceptable excipients vary according to the inhibitor used and the method of administration chosen. The methods and routes of administration of the inhibitors used according to the invention may be adapted by those skilled in the art according to the subject and the inhibitor used. The determination of the dose at which said inhibitor is used according to the invention may be performed using techniques known to those skilled in the art. This dose will be dependent on various factors comprising in particular the activity of the inhibitor, the method of administration, the duration of administration, the duration of the treatment, other medicinal products or compounds used in conjunction with the inhibitor, age, sex, weight, general health and previous medical history of the subject treated. Compositions which comprise a therapeutically-effective amount of one or more of the compounds are formulated together with one or more acceptable carriers (additives) and/or diluents. The compositions may contain one or more of wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. Compositions include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The compositions may be in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of

administration. Illustrating the invention are the following examples that are not to be considered as limiting the invention to their details. EXAMPLES Materials and Methods Materials All common reagents such as HEPES, MgCl₂ and dimethyl sulfoxide (DMSO) were reagent grade quality obtained from Thermo Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO). Compounds were purchased from Life Chemicals (Niagara-on-the-Lake ON, Canada), Enamine (Monmouth Jct., NJ), and Vitas M Labs (Narva, Estonia) or synthesized. Purchased hit compounds (#1– 3) were verified for purity and MW by HPLC-MS. Compound 1 was also re-synthesized. Compound synthesis

Synthesis of compound 1 and its analogs along with compounds 4, 5 and 5a are described in the“Design and Synthesis of MEKK2 Inhibitors” section. MEKK2 Biochemical Assays The MEKK2 intrinsic ATPase activity assay, MEKK2 TR-FRET binding assay and the transphosphorylation assay were performed as described above and in reference 31. MEKK2 activity assays were performed using ATP concentrations at the apparent Km of the assay format, unless otherwise noted. IC₅₀ value determinations IC₅₀ was defined as the concentration of inhibitor that generates a 50% reduction in assay signal. Compounds were dissolved at 10 mM in 100% DMSO to make stock solutions. Serial dilutions of compounds were performed in 100% DMSO then subsequently diluted into assay buffer and used in the MEKK2 enzymatic assay. For all MEKK2 assay formats, compound concentration response curves were generated using data points that represent the average of 2 or 3 determinations per concentration and 8-10 compound concentrations tested. All IC₅₀ values provided are averages of at least two independent determinations. The IC₅₀ values were calculated from concentration response data using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) employing either a four- or three-parameter (fixed bottom) curve fit. Selectivity Assessment by Kinase Profiling Panels Selected compounds were profiled for activity in serine/threonine and tyrosine kinase panels by Carna Biosciences as previously reported (32). In the 50-member kinase panel testing, compounds were tested at 1 mM concentration in duplicate kinase reactions and average percent inhibition reported. The external IC₅₀ determinations were performed using ELISA- based activity assays with a dose response where each concentration was tested in duplicate and data averaged before calculating the IC₅₀ values. As single-point selectivity testing or dose-response assays, external kinase assays were performed using ATP concentrations at the apparent Km of ATP in the kinase assay, unless otherwise noted. Design and Synthesis of MEKK2 Inhibitors

General Information. All commercial grade anhydrous solvents and reagents were used as received from vendors without further purification unless otherwise stated. All the reactions were performed in oven-dried glassware (either in round-bottom flasks or 25 ml vials fitted with rubber septa) under an atmosphere of nitrogen and the progress of reactions was monitored by thin-layer chromatography and GC/MS (EI) and/or HPLC/MS (ESI-APCI) analysis. Visualization was performed by ultraviolet light and/or by staining with phosphomolybdic acid (PMA) or p-anisaldehyde. All purifications were either carried by recrystallization from a DMSO–MeOH mixture, or by flash column chromatography on a CombiFlash^®R_f System using normal-phase disposable prepacked silica gel columns eluting with either EtOAc/hexane or MeOH/CH₂Cl₂ mixtures. Melting points are uncorrected. Data for ¹H and ¹³C NMR spectra were recorded on 500 MHz spectrometer. Chemical shifts (d) are reported in parts per million (ppm) using tetramethylsilane as an internal standard. Multiplicities are reported using the following abbreviations: br = broad; s = singlet; d = doublet; t = triplet; q = quartet; quint = quintet, sex = sextet, sep = septet, m = multiplet. Mass spectra were recorded on HPLC/MS instrument equipped with a XTerra_MS (C-18, 3.5mm) 3.0 × 100mm column. Representative Procedure for the synthesis of MEKK2 inhibitors 1b–s. Scheme 1

Step 1: An oven dried 25 mL glass round bottom flask was charged with 2-hydroxy-3- methoxy-benzaldehyde (3.000 g, 19.72 mmol), 2-cyanoacetamide (1.660 g, 19.74 mmol) and i-PrOH (4 mL), and the stirred mixture heated to 40–45 °C. Piperidine (0.12 mL, 1.21 mmol) was added and the resulting clear orange solution stirred for 10 min to reaction completion affording a greenish grey precipitate. Upon cooling to room temperature, the precipitate was collected, filter cake washed with i-PrOH (2 × 10 mL) then dried under reduced pressure to afford 2-imino-8-methoxy-2H-chromene-3-carboxamide (3.900 g, 91%) as a greenish grey amorphous solid (used in subsequent transformations without further purification).¹ Step 2: An oven dried 25 mL glass vial was charged with 2-imino-8-methoxy-2H-chromene- 3-carboxamide (0.100 g, 0.458 mmol), 4-amino-2-chlorophenol (0.066 g, 0.460 mmol) and glacial AcOH (4 mL), and the mixture stirred at 70°C for 45 min resulting in the formation of a precipitate.¹ The reaction mixture was cooled to room temperature, the precipitate collected, then washed with ice cold ethanol followed by drying under reduced pressure to afford orange solid contaminated by AcOH. Recrystallization from a DMSO–MeOH mixture gave pure 2-(3-chloro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b) (0.076 g, 48%) as golden yellow solid. 2-(3-Chloro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b). The product 1b was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-2-chlorophenol in 48% yield as golden yellow solid: mp 263-264 °C; ¹H NMR (CDCl₃, 500 MHz) ^G 3.93 (s, 3H), 6.97 (d, 1H, J = 8.5 Hz), 7.28–7.21 (m, 1H), 7.38–7.28 (m, 3H), 7.87 (d, 1H, J = 4.0 Hz), 7.98 (s, 1H), 8.43 (s, 1H), 9.42 (d, 1H, J = 3.5 Hz), 10.16 (s, 1H); APCI/ESI-MS: m/z 345.0 [M+H]⁺. 2-(4-Hydroxy-3-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1c). The product 1c was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-2-methylphenol in 46% yield as yellow solid: mp 238-240°C; ¹H NMR (CDCl₃, 500 MHz) G 2.18 (s, 3H), 3.92 (s, 3H), 6.79 (d, 1H, J = 8.5 Hz), 7.26–7.18 (m, 1H), 7.37–7.26 (m, 3H), 7.58 (s, 1H), 7.86 (br s, 1H), 8.37 (s, 1H), 9.34 (s, 1H), 9.62 (br s, 1H); APCI/ESI-MS: m/z 325.1 [M+H]⁺. 2-(3,4-Dihydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1d). The product 1d was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- aminobenzene-1,2-diol² in 12% yield as brown solid: mp 218-220 °C; ¹H NMR (CDCl₃, 500 MHz) G 3.89 (s, 3H), 6.74 (d, J = 8.0 Hz, 1H), 7.11–6.96 (m, 2H), 7.42–7.16 (m, 3H), 7.87 (br s, 1H), 8.37 (s, 1H), 8.82 (s, 1H), 8.97 (s, 1H), 9.58 (br s, 1H); APCI/ESI-MS: m/z 327.0 [M+H]⁺. 2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e). The product 1e was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-2-flurophenol in 57% yield as yellow solid: mp 266-268°C; ¹H NMR (CDCl₃, 500 MHz) G 3.91 (s, 3H), 7.01–6.89 (m, 1H), 7.41–7.17 (m, 4H), 7.65 (d, 1H, J = 14.0 Hz), 7.88 (s, 1H), 8.43 (s, 1H), 9.42 (d, 1H, J = 3.5 Hz), 9.84 (s, 1H); APCI/ESI-MS: m/z 329.1 [M+H]⁺. 2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f). The product 1f was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4-amino-3-(trifluoromethyl)phenol³ in 56% yield as canary yellow amorphous solid; mp 288-290 °C; ¹H NMR (CDCl₃, 500 MHz) G 3.82 (s, 3H), 7.14–7.02 (m, 2H), 7.34–7.20 (m, 2H), 7.37 (d, 1H, J = 7.5 Hz), 7.72 (d, 1H, J = 9.0 Hz), 8.00 (d, 1H, J = 3.0 Hz), 8.55 (s, 1H), 9.21 (d, 1H, J = 3.0 Hz), 9.95 (s, 1H); APCI/ESI-MS: m/z 379.1 [M+H]⁺. 2-(4-Hydroxy-2-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1g). The product 1g was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-3-methlphenol in 20% yield as golden yellow amorphous solid: mp 233-235 °C; ¹H NMR (CDCl₃, 500 MHz) G 2.18 (s, 3H), 3.82 (s, 3H), 6.61 (dd, 2H, J = 2.5, 8.5 Hz), 7.24– 7.19 (m, 1H), 7.26 (dd, 1H, J = 2.0, 8.5 Hz), 7.31 (dd, 1H, J = 2.0, 7.8 Hz), 7.50 (d, 1H, J = 8.5 Hz), 7.93 (d, 1H, J = 4.0 Hz), 8.40 (s, 1H), 9.23 (s, 1H), 9.58 (d, 1H, J = 4.0 Hz); APCI/ESI-MS: m/z 325.1 [M+H]⁺. 2-(2-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1h). The product 1h was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-3-fluorophenol in 63% yield as orange solid: mp 277-278 °C; ¹H NMR (CDCl₃, 500 MHz) G 3.90 (s, 3H), 6.72–6.62 (m, 2H), 7.42–7.20 (m, 3H), 7.85 (t, 1H, J = 9.5 Hz), 7.94 (br s, 1H), 8.47 (s, 1H), 9.63 (br s, 1H), 9.88 (s, 1H); APCI/ESI-MS: m/z 329.1 [M+H]⁺. 2-(2,3-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1i). The product 1i was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-2,3-difluorophenol in 64 % yield as yellow solid; mp 269-270 °C; ¹H NMR (CDCl₃, 500 MHz) G 3.89 (s, 3H), 6.82 (t, 1H, J = 9.0 Hz), 7.42–7.22 (m, 3H), 7.57 (t, 1H, J = 9.0 Hz), 7.97 (br s, 1H), 8.52 (s, 1H), 9.45 (br s, 1H), 10.34 (s, 1H); APCI/ESI-MS: m/z 347.0 [M+H]⁺. 2-(3,5-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1j). The product 1j was prepared from 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4- amino-2,6-difluorophenol⁴ in 40 % yield as yellow solid: mp >300°C; ¹H NMR (CDCl₃, 500 MHz) G 3.91 (s, 3H), 7.49–7.20 (m, 5H), 7.88 (d, 1H, J = 3.0 Hz), 8.47 (s, 1H), 9.26 (d, 1H, J = 2.5 Hz), 10.11 (s, 1H APCI/ESI-MS: m/z 347.0 [M+H]⁺. 2-(2,6-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1k). The product 1k was prepared 2-imino-8-methoxy-2H-chromene-3-carboxamide and 4-amino- 3,5-difluorophenol in 74 % yield as yellow solid: mp 280-281°C; ¹H NMR (CDCl₃, 500 MHz) G 3.77 (s, 3H), 6.55 (d, 2H, J = 9.5 Hz), 7.34–7.20 (m, 2H), 7.39 (d, 1H, J = 7.5 Hz), 7.97 (br s, 1H), 8.55 (s, 1H), 9.12 (br s, 1H), 10.10 (s, 1H); APCI/ESI-MS: m/z 347.0 [M+H]⁺. 8-Ethoxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1l). The intermediate, 8-ethoxy-2-imino-2H-chromene-3-carboxamide was prepared from 3-ethoxy-2- hydroxybenzaldehyde and 2-cyanoacetamide in 76% yield as yellow amorphous solid: mp 161-162 °C; ¹H NMR (CDCl₃, 500 MHz) G 1.39 (t, 3H, J = 11.5 Hz), 4.17 (q, 2H, J = 11.5 Hz), 7.21–7.08 (m, 1H), 7.40–7.21 (m, 2H,) 7.79 (br s, 1H), 8.37 (s, 1H), 8.93 (br s, 1H), 9.61 (br s, 1H); APCI/ESI-MS: m/z 233.0 [M+H]⁺. The product 1l was prepared from 8-ethoxy-2-imino-2H-chromene-3-carboxamide and 4- aminophenol in 63% yield as yellow solid: mp 265-267°C; ¹H NMR (CDCl₃, 500 MHz) G 1.43 (t, 3H, J = 7.0 Hz), 4.14 (q, 2H, J = 7.0 Hz), 6.77 (d, 2H, J = 9.0 Hz), 7.34–7.217 (m, 3H), 7.60 (d, 2H, J = 8.5 Hz), 7.88 (d, 1H, J = 3.5 Hz), 8.39 (s, 1H), 9.46 (s, 1H), 9.61 (d, 1H, J = 4.0 Hz); APCI/ESI-MS: m/z 325.1 [M+H]⁺. 2-(4-Hydroxyphenylimino)-8-propoxy-2H-chromene-3-carboxamide (1m). The intermediate, 2-imino-8-propoxy-2H-chromene-3-carboxamide was prepared from 2- hydroxy-3-propoxybenzaldehyde and 2-cyanoacetamide in 52% yield as white solid: mp 168–170ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 1.01 (t, 3H, J = 8.0 Hz), 1.78 (sex, 2H, J = 8.0 Hz), 4.06 (t, 2H, J = 8.0 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.26 (dd, 2H, J = 8.5, 13.0 Hz), 7.77 (s, 1H), 8.36 (s, 1H), 8.90 (s, 1H), 9.56 (s, 1H); APCI/ESI-MS: m/z 247.1 [M+H]⁺. The product 1m was prepared from 2-imino-8-propoxy-2H-chromene-3-carboxamide and 4- aminophenol in 80% yield as yellow solid: mp 283–285 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 1.07 (t, 3H, J = 7.0 Hz), 1.82 (sex, 2H, J = 6.0 Hz), 4.05 (t, 2H, J = 6.0 Hz), 6.76 (d, 2H, J = 8.5 Hz), 7.20 (t, 1H, J = 8.0 Hz), 7.26 (d, 1H, J = 8.0 Hz), 7.29 (d, 1H, J = 8.0 Hz), 7.60 (d, 2H, J = 8.5 Hz), 7.88 (d, 1H, J = 3.5 Hz), 8.38 (s, 1H), 9.46 (s, 1H), 9.60 (d, 1H, J = 4.5 Hz); APCI/ESI-MS: m/z 339.1 [M+H]⁺. 2-(4-Hydroxyphenylimino)-8-isopropoxy-2H-chromene-3-carboxamide (1n). The intermediate, 2-imino-8-isopropoxy-2H-chromene-3-carboxamide was prepared from 2- hydroxy-3-isopropoxybenzaldehyde and 2-cyanoacetamide in 75% yield as white solid: mp 182–184 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 1.31 (d, 6H, J = 6.5 Hz), 1.78 (sep, 1H, J = 6.5 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.27 (dd, 2H, J = 8.0, 12.5 Hz), 7.76 (s, 1H), 8.36 (s, 1H), 8.87 (s, 1H), 9.58 (s, 1H); APCI/ESI-MS: m/z 247.1 [M+H]⁺. The product 1n was prepared from and 2-imino-8-isopropoxy-2H-chromene-3-carboxamide and 4-aminophenol in 80% yield as yellow solid: mp 254–256 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 1.32 (d, 6H, J = 6.0 Hz), 4.69 (sep, 1H, J = 6.0 Hz), 6.77 (d, 2H, J = 8.5 Hz), 7.19 (t, 1H, J = 7.5 Hz), 7.30 (dd, 2H, J = 6.0, 7.5 Hz), 7.58 (d, 2H, J = 8.5 Hz), 7.87 (d, 1H, J = 3.5 Hz), 8.38 (s, 1H), 9.45 (s, 1H), 9.59 (d, 1H, J = 3.5 Hz); APCI/ESI-MS: m/z 339.1 [M+H]⁺. 8-Fluoro-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1o). The intermediate 8-fluoro-2-imino-2H-chromene-3-carboxamide, prepared from 3-fluoro-2- hydroxybenzaldehyde and 2-cyanoacetamide, was contaminated with some impurities and the attempted purification failed to give pure one. Hence, it was carried forward as such to the next step for the preparation of 1o by reacting with 4-aminophenol in 5% yield as yellow solid: mp 276–278 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 6.76–6.80 (m, 2H), 7.25 (dt, 1H, J = 4.5, 8.0 Hz), 7.31–7.35 (m, 2H), 7.50 (ddd, 1H, J = 1.5, 8.5, 11.0 Hz), 7.58 (d, 1H, J = 7.5 Hz), 7.91 (d, 1H, J = 3.5 Hz), 8.42 (d, 1H, J = 2.0 Hz), 9.36 (d, 1H, J = 3.5 Hz), 9.45 (s, 1H); ¹³C NMR (125 MHz) d 115.2, 118.8 (d, J = 16.7 Hz), 120.9, 123.3, 124.3 (d, J = 7.0 Hz), 124.9, 125.5, 134.8, 138.9, 140.6 (d, J = 11.1 Hz), 145.3, 147.2, 149.2, 155.0, 162.6; APCI/ESI-MS: m/z 299.1 [M+H]⁺. 8-Bromo-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1p). The intermediate, 8-bromo-2-imino-2H-chromene-3-carboxamide was prepared from 3-bromo-2- hydroxybenzaldehyde and 2-cyanoacetamide in 63% as yellow solid: mp 190–192 ºC; ¹H NMR (DMSO-d₆+CDCl_3, 500 MHz) d 6.85 (br s, 1H), 7.11 (t, 1H, J = 8.0 Hz), 7.47 (dd, 1H, J = 1.5, 7.5 Hz), 7.68 (dd, 1H, J = 1.5, 8.0 Hz), 8.08 (s, 1H), 8.39 (d, 1H, J = 1.5 Hz), 9.72 (br s, 1H); ¹³C (125 MHz) d 108.6, 120.1, 121.5, 124.8, 128.7, 135.9, 141.3, 150.6, 156.0, 163.3; APCI/ESI-MS: m/z 266.9 [M+H]⁺. The product 1p was prepared from 8-bromo-2-imino-2H-chromene-3-carboxamide and 4- aminophenol in 82% yield as orange solid: mp 263–265 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 6.79 (d, 2H, J = 8.0 Hz), 7.21 (t, 1H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.50 (d, 1H, J = 7.5 Hz), 7.81 (d, 1H, J = 8.0 Hz), 7.91 (d, 1H, J = 3.5 Hz), 8.38 (s, 1H), 9.36 (d, 1H, J = 3.5 Hz), 9.45 (s, 1H); ¹³C NMR (125 MHz) d 107.7, 115.1, 120.6, 123.2, 125.4, 126.0, 129.0, 134.5, 135.5, 139.1, 145.3, 149.8, 155.1, 162.6; APCI/ESI-MS: m/z 359.0 [M+H]⁺. 8-Hydroxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1q). The intermediate, 8-hydroxy-2-imino-2H-chromene-3-carboxamide was unstable on isolation. Hence, the solvent (n-BuOH) was removed under reduced pressure and the crude intermediate was carried forward to the next step to give the product 1q in 8% yield (one pot, 2 steps) as brown solid, after flash silica gel column chromatography using MeOH–CH₂Cl₂ (1:9): mp 235–237 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 6.76 (d, 2H, J = 8.0 Hz), 7.08 (d, 2H, J = 5.0 Hz), 7.17 (d, 1H, J = 4.0 Hz), 7.51 (d, 2H, J = 8.0 Hz), 7.86 (d, 1H, J = 0.5 Hz), 8.36 (s, 1H), 9.43 (s, 1H), 9.60 (d, 1H, J = 0.5 Hz), 10.16 (s, 1H); ¹³C NMR (125 MHz) d 115.1, 119.1, 119.4, 119.6, 124.2, 126.2, 140.1, 141.5, 144.2, 155.0, 163.0; APCI/ESI-MS: m/z 297.1 [M+H]⁺.

8-Ethoxy-2-(4-hydroxy-2-(trifluoromethyl)phenylimino)-2H-chromene-3-carboxamide (1r). The product 1r was prepared from 8-ethoxy-2-imino-2H-chromene-3-carboxamide and 4-amino-3-(trifluoromethyl)phenol in 67% yield as yellow solid: mp 265-266 °C; ¹H NMR (CDCl₃, 500 MHz) G 1.33 (t, 3H, J = 7.0 Hz), 4.07 (q, 2H, J = 7.0 Hz), 7.04 (dd, 1H, J = 2.5, 8.5 Hz), 7.11 (d, 1H, J = 2.5 Hz), 7.29–7.19 (m, 2H), 7.35 (d, 1H, J = 7.5 Hz), 7.90 (d, 1H, J = 9.0 Hz), 8.02 (d, 1H, J = 3.5 Hz), 8.54 (s, 1H), 9.24 (d, 1H, J = 3.5 Hz), 9.97 (s, 1H); APCI/ESI-MS: m/z 393.1 [M+H]⁺. 8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). The product 1s was prepared from 8-ethoxy-2-imino-2H-chromene-3-carboxamide and 4-amino- 3-methylphenol in 47% yield as orange solid: mp 255-256 °C; ¹H NMR (CDCl₃, 500 MHz) G 1.35 (t, 3H, J = 7.0 Hz); 2.21 (s, 3H), 4.08 (q, 2H, J = 7.0 Hz), 6.60 (dd, 1H, J = 3.0, 8.5 Hz), 6.68 (d, 1H, J = 2.5 Hz), 7.22–7.16 (m, 1H), 7.24 (dd, 1H, J = 1.5, 8.0 Hz), 7.31 (dd, 1H, J = 1.5, 7.5 Hz), 7.73 (d, 1H, J = 8.5 Hz), 7.94 (d, 1H, J = 4.5 Hz), 8.40 (s, 1H), 9.26 (s, 1H), 9.63 (d, 1H, J = 4.0 Hz); APCI/ESI-MS: m/z 339.1 [M+H]⁺. Scheme 2

2-(4-Hydroxyphenylamino)-8-methoxyquinoline-3-carboxamide (4). Iron powder (5.000 g, 89.525 mmol) and conc. HCl (0.2 mL) were added to a solution of 3-methoxy-2- nitrobenzaldehyde (10) (2.000 g, 11.041 mmol) in a solvent mixture of EtOH and water (40 mL, 4:1), and the mixture was heated at reflux for 2 h. After allowing the mixture to cool to room temperature, it was filtered through a pad of Celite and washed well with EtOAc (60 mL). The filtrate was evaporated under reduced pressure, and the residue was purified by flash silica gel column chromatography (Combiflash Rf) using EtOAc–hexanes (1:4) to afford 2-amino-3-methoxybenzaldehyde⁵ (1.420 g, 85%) as a yellow syrup: ¹H NMR (CDCl_3, 500 MHz) d 3.88 (s, 3H), 6.83 (br s, 2H), 6.68 (t, 1H, J = 8.0 Hz), 6.88 (dd, 1H, J = 1.0, 8.0 Hz), 7.12 (dd, 1H, J = 1.5, 8.0 Hz), 7.26 (s, 1H), 9.89 (s, 1H); APCI/ESI-MS: m/z 152.1 [M+H]⁺. To a solution of 2-amino-3-methoxybenzaldehyde (1.400 g, 9.262 mmol) in absolute EtOH (16 mL) were added dimethyl malonate (3.2 mL, 28.001 mmol) followed by glacial AcOH (0.1 mL) and piperidine (2.75 mL, 27.839 mmol), and the mixture refluxed for 3 h. After allowing the reaction mixture reaching room temperature, it was diluted with water (15 mL) and extracted with EtOAc (50 mL), washed with brine, dried and evaporated under reduced pressure. The residue was triturated in EtOAc/Et₂O and the resulting solid was collected and dried to give the pure methyl 8-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (1.800 g, 83%) as yellow solid: mp 146–148ºC; ¹H NMR (CDCl_3, 500 MHz) d 3.96 (s, 3H), 3.99 (s, 3H), 7.05 (d, 1H, J = 7.5 Hz), 7.17 (t, 1H, J = 7.5 Hz), 7.24 (d, 1H, J = 7.5 Hz), 8.54 (s, 1H), 9.26 (br s, 1H); APCI/ESI-MS: m/z 234.1 [M+H]⁺. A neat POCl₃ (8 mL) was added to 8-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (1.800 g, 7.718 mmol) and refluxed for 3 h. After cooling the mixture to room temperature, the excess POCl₃ was evaporated under reduced pressure and the residue dissolved in CH₂Cl_2. The mixture was carefully neutralized with 2M NaOH, extracted with CH₂Cl₂ (40 mL), washed with brine, dried and evaporated under reduced pressure to give methyl 2-chloro-8- methoxyquinoline-3-carboxylate⁶ (1.900 g, 98%) as a light yellow solid: mp 66–68 ºC; ¹H NMR (CDCl_3, 500 MHz) d 4.01 (s, 3H), 4.08 (s, 3H), 7.18 (d, 1H, J = 8.0 Hz), 7.45 (d, 1H, J = 8.0 Hz), 7.55 (t, 1H, J = 8.0 Hz), 8.65 (s, 1H); APCI/ESI-MS: m/z 252.0 [M+H]⁺. To a solution of methyl 2-chloro-8-methoxyquinoline-3-carboxylate (0.340 g, 1.351 mmol) in absolute EtOH (3 mL) was added 4-aminophenol (0.160 g, 1.466 mmol) and heated at 90ºC for 12 h.⁷ After completion of the reaction (as judged by tlc and LCMS), ethanol was evaporated under reduced pressure and the residue purified by flash silica gel column chromatography (Combiflash Rf) using MeOH–CH₂Cl₂ (1:19) to afford methyl 2-(4- hydroxyphenylamino)-8-methoxyquinoline-3-carboxylate (0.380 g, 80%) as yellow solid: mp 192–194 ºC; ¹H NMR (CDCl_3, 500 MHz) d 4.00 (s, 3H), 4.04 (s, 3H), 4.54 (s, 1H), 6.86 (d, 2H, J = 8.5 Hz), 7.04 (d, 1H, J = 8.0 Hz), 7.19 (t, 1H, J = 8.0 Hz), 7.27 (d, 1H, J = 8.0 Hz), 7.94 (d, 2H, J = 8.5 Hz), 8.74 (s, 1H), 10.15 (s, 1H); APCI/ESI-MS: m/z 325.1 [M+H]⁺.

To a solution of methyl 2-(4-hydroxyphenylamino)-8-methoxyquinoline-3-carboxylate (0.130 g, 0.401 mmol) in 2M NH₃ in MeOH (10 mL) was added 14M NH₄OH (10 mL) and stirred at room temperature for 12 h.⁸ After completion of the reaction (as judged by tlc and LCMS), it was carefully neutralized with 3M HCl and extracted with EtOAc (2 x 20 mL), washed with brine, dried over Na₂SO₄, evaporated under reduced pressure, and the residue purified by flash silica gel column chromatography (Combiflash Rf) using MeOH–CH2Cl2 (1:19) to afford 4 (0.080 g, 64%) as a yellow solid: mp 258–260 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 3.95 (s, 3H), 6.74 (d, 2H, J = 8.0 Hz), 7.15 (d, 1H, J = 7.5 Hz), 7.22 (t, 1H, J = 7.5 Hz), 7.30 (d, 1H, J = 7.5 Hz), 7.81–7.86 (m, 3H), 8.45 (s, 1H), 8.60 (s, 1H), 9.04 (s, 1H), 10.79 (s, 1H); APCI/ESI-MS: m/z 310.1 [M+H]⁺. Scheme 3

2-(4-Hydroxyphenoxy)-8-methoxyquinoline-3-carboxamide (5). Sodium hydride (0.080 g, 3.333 mmol) was slowly added in portions to a solution of hydroquinone (0.180 g, 1.635 mmol) in anhydrous THF (4 mL) at 0ºC and stirred for 15 min. Then, a solution of methyl 2- chloro-8-methoxyquinoline-3-carboxylate (0.200 g, 0.795 mmol) in anhydrous THF (2 mL) was added and heated at 60 ºC for 24 h. After completion, the reaction mixture was quenched by adding 1M HCl, extracted with EtOAc (2 x 40 mL), washed with brine, dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by flash silica gel column chromatography (Combiflash Rf) using EtOAc–hexanes (1:3) to afford methyl 2-(4- hydroxyphenoxy)-8-methoxyquinoline-3-carboxylate (0.140 g, 54%) as off-white solid: mp 234–236 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 3.81 (s, 3H), 3.91 (s, 3H), 6.78–6.82 (m, 2H), 7.02–7.05 (m, 2H), 7.24 (d, 1H, J = 8.0 Hz), 7.45 (t, 1H, J = 8.0 Hz), 7.62 (d, 1H, J = 8.0 Hz), 8.83 (s, 1H), 9.34 (s, 1H); APCI/ESI-MS: m/z 326.1 [M+H]⁺. The product 5 was prepared from methyl 2-(4-hydroxyphenoxy)-8-methoxyquinoline-3- carboxylate using 14M NH4OH and 2M NH3 in MeOH (1:1) in 33% yield as off-white solid: mp 264–266 ºC; ¹H NMR (DMSO-d_6, 500 MHz) d 3.79 (s, 3H), 6.78–6.82 (m, 2H), 7.07– 7.11 (m, 2H), 7.18 (dd, 1H, J = 1.0, 8.0 Hz), 7.41 (t, 1H, J = 8.0 Hz), 7.57 (dd, 1H, J = 1.0, 8.0 Hz), 7.78 (br s, 1H), 7.90 (br s, 1H), 8.63 (s, 1H), 9.35 (s, 1H); APCI/ESI-MS: m/z 311.1 [M+H]⁺.

Scheme 4

8-Ethoxy-2-(4-hydroxy-2-methylphenoxy)quinoline-3-carboxamide (5a). To a solution of 3-hydroxy-2-nitrobenzaldehyde (2.000 g, 11.967mmol) in anhydrous DMF (20 mL) were added K₂CO₃ (5.000 g, 36.177 mmol) and Ethyl iodide (2.9 mL, 36.071 mmol) at room temperature and then heated at 80 ºC for 12 h. The reaction mixture was diluted with water (20 mL), extracted with EtOAc (80 mL) washed with brine, dried over Na₂SO₄, evaporated under reduced pressure, and the residue purified by flash silica gel column chromatography (Combiflash Rf) using EtOAc–hexanes (1:4) to afford 3-ethoxy-2-nitrobenzaldehyde (2.000 g, 86%) as white solid: mp 68–70 ºC; ¹H NMR (CDCl_3, 500 MHz) d 1.44 (t, 3H, J = 7.0 Hz), 4.19 (q, 2H, J = 7.0 Hz), 7.32 (dd, 1H, J = 1.0, 7.5 Hz), 7.49 (dd, 1H, J = 1.0, 7.5 Hz), 7.59 (t, 1H, J = 7.5 Hz), 9.94 (s, 1H); ¹³C NMR (125 MHz) d 14.4, 65.8, 119.4, 121.9, 131.5, 150.5, 186.9; APCI/ESI-MS: m/z 196.0 [M+H]⁺. 2-Amino-3-ethoxybenzaldehyde was prepared from 3-ethoxy-2-nitrobenzaldehyde using Fe(0) powder and conc. HCl in EtOH-water mixture in 56% yield as light yellow syrup: ¹H NMR (CDCl_3, 500 MHz) d 1.45 (t, 3H, J = 7.0 Hz), 4.08 (q, 2H, J = 7.0 Hz), 6.40 (br s, 2H), 6.66 (t, 1H, J = 7.5 Hz), 6.86 (d, 1H, J = 7.5 Hz), 7.10 (dd, 1H, J = 1.0, 7.5 Hz), 9.88 (s, 1H); ¹³C NMR (125 MHz) d 14.8, 64.1, 114.6, 115.0, 118.2, 126.4, 141.0, 145.9, 193.9; APCI/ESI-MS: m/z 166.1 [M+H]⁺. Ethyl 8-ethoxy-2-oxo-1,2-dihydroquinoline-3-carboxylate was prepared from 2-amino-3- ethoxybenzaldehyde using diethyl malonate, AcOH, and piperidine in absolute EtOH⁹ in 92% yield as orange solid: mp 98–100 ºC; ¹H NMR (CDCl_3, 500 MHz) d 1.42 (t, 3H, J = 7.0 Hz), 1.52 (t, 3H, J = 7.0 Hz), 4.20 (q, 2H, J = 7.0 Hz), 4.43 (q, 2H, J = 7.0 Hz), 7.03 (d, 1H, J = 8.0 Hz), 7.15 (t, 1H, J = 8.0 Hz), 7.23 (d, 1H, J = 8.0 Hz), 8.49 (s, 1H), 9.26 (br s, 1H); ¹³C NMR (125 MHz) d 14.3, 14.7, 61.5, 64.6, 112.5, 118.5, 120.5, 122.5, 123.3, 130.1, 144.4, 145.2, 158.6, 164.3; APCI/ESI-MS: m/z 262.1 [M+H]⁺. The product ethyl 2-chloro-8-ethoxyquinoline-3-carboxylate was prepared from ethyl 8- ethoxy-2-oxo-1,2-dihydroquinoline-3-carboxylate using POCl₃ in 89% yield as white solid: mp 66–68 ºC; ¹H NMR (CDCl_3, 500 MHz) d 1.46 (t, 3H, J = 7.0 Hz), 1.60 (t, 3H, J = 7.0 Hz), 4.33 (q, 2H, J = 7.0 Hz), 4.46 (q, 2H, J = 7.0 Hz), 7.17 (d, 1H, J = 8.0 Hz), 7.44 (dd, 1H, J = 1.0, 8.0 Hz), 7.52 (t, 1H, J = 8.0 Hz), 8.62 (s, 1H); ¹³C NMR (125 MHz) d 14.2, 14.4, 62.1, 64.7, 111.7, 119.6, 125.1, 127.1, 128.1, 140.1, 141.3, 146.6, 153.8, 164.7; APCI/ESI-MS: m/z 280.0 [M+H]⁺. To a solution of Ethyl 2-chloro-8-ethoxyquinoline-3-carboxylate (0.100 g, 0.357 mmol) in anhydrous DMF (4 mL) were added 4-(methoxymethoxy)-2-methylphenol¹⁰ (0.090 g, 0.535 mmol) and Cs₂CO₃ (0.240 g, 0.737 mmol), and heated at 80 ºC for 12 h. After completion, water (10 mL) was added to the mixture, extracted with EtOAc (30 mL), washed with brine, dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by flash silica gel column chromatography (Combiflash Rf) using EtOAc–hexanes (1:9) to afford ethyl 8-ethoxy-2-(4-(methoxymethoxy)-2-methylphenoxy)quinoline-3-carboxylate (0.125 g, 85%) as white solid: mp 70–72ºC; ¹H NMR (CDCl_3, 500 MHz) d 1.26 (t, 3H, J = 7.0 Hz), 1.44 (t, 3H, J = 7.0 Hz), 2.21 (s, 3H), 3.50 (s, 3H), 4.05 (q, 2H, J = 7.0 Hz), 4.46 (q, 2H, J = 7.0 Hz), 5.18 (s, 2H), 6.91 (dd, 1H, J = 2.5, 8.5 Hz), 6.95 (d, 1H, J = 8.0 Hz), 7.11 (dd, 1H, J = 1.5, 7.5 Hz), 7.24 (d, 1H, J = 8.5 Hz), 7.32 (t, 1H, J = 8.0 Hz), 7.43 (dd, 1H, J = 1.0, 8.0 Hz), 8.67 (s, 1H); APCI/ESI-MS: m/z 412.2 [M+H]⁺. 8-Ethoxy-2-(4-(methoxymethoxy)-2-methylphenoxy)quinoline-3-carboxamide was prepared from 8-ethoxy-2-(4-(methoxymethoxy)-2-methylphenoxy)quinoline-3-carboxylate using 14M NH₄OH and 2M NH₃ in MeOH (1:1) in 91% yield as white solid: mp 128–130 ºC; ¹H NMR (CDCl_3, 500 MHz) d 1.22 (t, 3H, J = 7.0 Hz), 2.16 (s, 3H), 3.51 (s, 3H), 4.02 (q, 2H, J = 7.0 Hz), 5.19 (s, 2H), 5.87 (br s, 1H), 6.96 (dd, 1H, J = 3.0, 8.5 Hz), 6.99 (d, 1H, J = 8.0 Hz), 7.11 (dd, 1H, J = 1.0, 7.5 Hz), 7.18 (d, 1H, J = 8.5 Hz), 7.34 (t, 1H, J = 8.0 Hz), 7.51 (dd, 1H, J = 1.0, 8.0 Hz), 8.02 (br s, 1H), 9.11 (s, 1H); APCI/ESI-MS: m/z 383.1 [M+H]⁺. To a suspension of 8-ethoxy-2-(4-(methoxymethoxy)-2-methylphenoxy)quinoline-3- carboxamide (0.150 g, 0.392 mmol) in anhydrous MeOH (5 mL) was added p-TSA (0.010 g, 0.058 mmol) and heated at 40 ºC for 12 h. After completion of the reaction (as judged by tlc and LCMS), the solvent was evaporated under reduced pressure, and the residue purified by flash silica gel column chromatography (Combiflash Rf) using MeOH–CH₂Cl₂ (1:19) to afford a white solid (0.090 g) which was a mixture of product and methyl hydroquinone (1:2). The mixture (in a round bottom flask) was dissolved in MeOH–CH₂Cl₂ (1:4), washed with sat. NaHCO₃ solution twice and neutralized with 1M aqueous HCl solution. The product was crashed out from the solvent mixture which was collected by filtration, washed with CH₂Cl₂ and collected to furnish 5a (0.050 g, 38%) as white solid: mp 220–222 ºC; ¹H NMR (DMSO- d_6, 500 MHz) d 1.14 (t, 3H, J = 7.0 Hz), 2.03 (s, 3H), 4.01 (q, 2H, J = 7.0 Hz), 6.62 (dd, 1H, J = 2.5, 8.5 Hz), 6.69 (d, 1H, J = 2.5 Hz), 7.05 (d, 1H, J = 8.5 Hz), 7.17 (d, 1H, J = 8.0 Hz), 7.36 (t, 1H, J = 8.0 Hz), 7.60 (d, 1H, J = 8.0 Hz), 7.80 (br s, 1H), 7.91 (br s, 1H), 8.67 (s, 1H), 9.23 (br s, 1H); APCI/ESI-MS: m/z 339.1 [M+H]⁺. Whereas particular features of the present invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details of the coating composition, coating, and methods disclosed herein may be made without departing from the scope in the appended claims.

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Claims

CLAIMS Claim 1. A method for inhibiting MEKK2 comprising administering a compound

wherein R’₁ is selected from hydrogen, fluorine, CF3, and methyl;

R’₂ is selected from hydrogen, chlorine, fluorine, hydroxyl and methyl;

R’₃ is selected from hydroxy and methoxy;

R’₄ and R’₅ are selected from hydrogen and fluorine; and

R is selected from methoxy, ethoxy, propoxy, isopropoxy. Claim 2. The method according to claim 1 wherein the compound is selected from

Claim 3. The method according to claim 1 wherein the compound is selected from 2-(3- Chloro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b);

2-(4-Hydroxy-3-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1c);

2-(3,4-Dihydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1d);

2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f);

2-(4-Hydroxy-2-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1g);

2-(2-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1h);

2-(2,3-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1i); 2- (3,5-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1j); 2-(2,6- Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1k); 8-Ethoxy-2- (4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1l);

2-(4-Hydroxyphenylimino)-8-propoxy-2H-chromene-3-carboxamide (1m); 2-(4- Hydroxyphenylimino)-8-isopropoxy-2H-chromene-3-carboxamide (1n);

8-Fluoro-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1o);

8-Bromo-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1p);

8-Hydroxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1q);

8-Ethoxy-2-(4-hydroxy-2-(trifluoromethyl)phenylimino)-2H-chromene-3-carboxamide (1r); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 4. The method according to claim 3 wherein the compound is selected from 2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 5. A method for inhibiting MEKK2 comprising administering a compound selected from

Claim 6. The method according to anyone of claims 1 to 5 wherein the compound is administered to a subject in need thereof. Claim 7. A compound

wherein R’₁ is selected from hydrogen, fluorine, CF3, and methyl;

R’₂ is selected from hydrogen, chlorine, fluorine, hydroxyl and methyl;

R’₃ is selected from hydroxy and methoxy;

R’₄ and R’₅ are selected from hydrogen and fluorine; and

R is selected from methoxy, ethoxy, propoxy, isopropoxy for inhibiting MEKK2. Claim 8. The compound according to claim 7 wherein the compound is selected from

Claim 9. The compound according to claim 7 wherein the compound is selected from 2- (3-Chloro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b);

2-(4-Hydroxy-3-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1c);

2-(3,4-Dihydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1d);

2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f);

2-(4-Hydroxy-2-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1g);

2-(2-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1h);

2-(2,3-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1i); 2- (3,5-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1j); 2-(2,6- Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1k); 8-Ethoxy-2- (4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1l); 2-(4-Hydroxyphenylimino)-8-propoxy-2H-chromene-3-carboxamide (1m); 2-(4- Hydroxyphenylimino)-8-isopropoxy-2H-chromene-3-carboxamide (1n);

8-Fluoro-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1o);

8-Bromo-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1p);

8-Hydroxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1q);

8-Ethoxy-2-(4-hydroxy-2-(trifluoromethyl)phenylimino)-2H-chromene-3-carboxamide (1r) and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 10. The compound according to claim 9 wherein the compound is selected from 2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 11. A compound for inhibiting MEKK2 selected from

Claim 12. Use of a compound of the formula

wherein R’₁ is selected from hydrogen, fluorine, CF3, and methyl;

R’₂ is selected from hydrogen, chlorine, fluorine, hydroxyl and methyl;

R’₃ is selected from hydroxy and methoxy;

R’₄ and R’₅ are selected from hydrogen and fluorine; and R is selected from methoxy, ethoxy, propoxy, isopropoxy for inhibiting MEKK2. Claim 13. Use of the compound according to claim 12 wherein the compound is selected from

Claim 14. Use of the compound according to claim 12 wherein the compound is selected from 2-(3-Chloro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b); 2-(4-Hydroxy-3-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1c);

2-(3,4-Dihydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1d);

2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f);

2-(4-Hydroxy-2-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1g);

2-(2-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1h);

2-(2,3-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1i); 2- (3,5-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1j); 2-(2,6- Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1k); 8-Ethoxy-2- (4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1l);

2-(4-Hydroxyphenylimino)-8-propoxy-2H-chromene-3-carboxamide (1m); 2-(4- Hydroxyphenylimino)-8-isopropoxy-2H-chromene-3-carboxamide (1n);

8-Fluoro-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1o);

8-Bromo-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1p);

8-Hydroxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1q);

8-Ethoxy-2-(4-hydroxy-2-(trifluoromethyl)phenylimino)-2H-chromene-3-carboxamide (1r) and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s) for inhibiting MEKK2. Claim 15. Use of the compound according to claim 14 wherein the compound is selected from 2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e); 2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 16. Use of a compound for inhibiting MEKK2 selected from

Claim 17. A compound of formula

wherein R’₁ is selected from hydrogen, fluorine, CF3, and methyl;

R’₂ is selected from hydrogen, chlorine, fluorine, hydroxyl and methyl;

R’₃ is selected from hydroxy and methoxy;

R’₄ and R’₅ are selected from hydrogen and fluorine; and

R is selected from methoxy, ethoxy, propoxy, isopropoxy. Claim 18. The compound according to claim 17 selected from 2-(3-Chloro-4- hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1b);

2-(4-Hydroxy-3-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1c);

2-(3,4-Dihydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1d);

2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f); 2-(4-Hydroxy-2-methylphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1g);

2-(2-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1h);

2-(2,3-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1i); 2- (3,5-Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1j); 2-(2,6- Difluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1k); 8-Ethoxy-2- (4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1l);

2-(4-Hydroxyphenylimino)-8-propoxy-2H-chromene-3-carboxamide (1m); 2-(4- Hydroxyphenylimino)-8-isopropoxy-2H-chromene-3-carboxamide (1n);

8-Fluoro-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1o);

8-Bromo-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1p);

8-Hydroxy-2-(4-hydroxyphenylimino)-2H-chromene-3-carboxamide (1q);

8-Ethoxy-2-(4-hydroxy-2-(trifluoromethyl)phenylimino)-2H-chromene-3-carboxamide (1r); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 19. A compound according to claim 18 selected from

2-(3-Fluoro-4-hydroxyphenylimino)-8-methoxy-2H-chromene-3-carboxamide (1e);

2-(4-Hydroxy-2-(trifluoromethyl)phenylimino)-8-methoxy-2H-chromene-3-carboxamide (1f); and

8-Ethoxy-2-(4-hydroxy-2-methylphenylimino)-2H-chromene-3-carboxamide (1s). Claim 20. A compound selected from