First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Small-molecule inhibition of MAP2K4 is synergistic with RAS inhibitors in KRAS-mutant cancers.

The Kirsten rat sarcoma viral oncogene homologue KRAS is among the most commonly mutated oncogenes in human cancers, thus representing an attractive target for precision oncology. The approval for clinical use of the first selective inhibitors of G12C mutant KRAS therefore holds great promise for cancer treatment. However, despite initial encouraging clinical results, the overall survival benefit that patients experience following treatment with these inhibitors has been disappointing to date, pointing toward the need to develop more powerful combination therapies. Here, we show that responsiveness to KRASG12C and pan-RAS inhibitors in KRAS-mutant lung and colon cancer cells is limited by feedback activation of the parallel MAP2K4-JNK-JUN pathway. Activation of this pathway leads to elevated expression of receptor tyrosine kinases that reactivate KRAS and its downstream effectors in the presence of drug. We find that the combination of sotorasib, a drug targeting KRASG12C, and the MAP2K4 inhibitor HRX-0233 prevents this feedback activation and is highly synergistic in a panel of KRASG12C-mutant lung and colon cancer cells. Moreover, combining HRX-0233 and sotorasib is well-tolerated and resulted in durable tumor shrinkage in mouse xenografts of human lung cancer cells, suggesting a therapeutic strategy for KRAS-driven cancers.

A Diverse and Versatile Regiospecific Synthesis of Tetrasubstituted Alkylsulfanylimidazoles as p38α Mitogen-Activated Protein Kinase Inhibitors.

An alternative strategy for the synthesis of 1-aryl- and 1-alkyl-2-methylsulfanyl-4-(4-fluorophenyl)-5-(pyridin-4-yl)imidazoles as potential p38α mitogen-activated protein kinase inhibitors is reported. The regioselective N-substitution of the imidazole ring was achieved by treatment of α-aminoketones with different aryl or alkyl isothiocyanates. In contrast to previously published synthesis routes starting from 2-amino-4-methylpyridine, the presented route is characterized by a higher flexibility and a lower number of steps. This strategy was also applied to access 1-alkyl-2-methylsulfanyl-5-(4-fluorophenyl)-4-(pyridin-4-yl)imidazoles in six steps starting from 2-chloro-4-methylpyridine.

Exploring the Molecular-Level Architecture of the Active Compounds in Liquisolid Drug Delivery Systems Based on Mesoporous Silica Particles: Old Tricks for New Challenges.

A general, easy-to-implement strategy for mapping the structure of organic phases integrated in mesoporous silica drug delivery devices is presented. The approach based on a few straightforward solid-state NMR techniques has no limitations regarding concentrations of the active compounds and enables straightforward discrimination of various organic phases. This way, among a range of typical arrangements of the active compounds and solvent molecules, a unique, previously unknown organogel phase of the self-assembled tapentadol in glucofurol as a solvent was unveiled and clearly identified. Subsequently, with an aid of 2D 1H-1H MAS NMR and high-level quantum-chemical calculations this uncommon low-molecular-weight organogel phase, existing exclusively in the porous system of the silica carrier, was described in detail. The optimized model revealed the tendency of tapentadol molecules to form hydrophobic arrangements through -OH···π interactions combined with π-π stacking occurring in the core of API aggregates, thus precluding the formation of hydrogen bonds with the solvent. Overall, the proposed experimental approach allows for clear discrimination of a variety of local structures of active compounds loaded in mesoporous silica drug delivery devices in reasonably short time being applicable for advancement of novel drug delivery systems in pharmaceutical industry.

From 2-Alkylsulfanylimidazoles to 2-Alkylimidazoles: An Approach towards Metabolically More Stable p38α MAP Kinase Inhibitors.

In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine) undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38α mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group resulted in 2-alkylimidazole derivatives 1 and 2, respectively, having a remarkably improved metabolic stability. The 2-alkylimidazole analogs 1 and 2 showed 20% and 10% biotransformation after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold increased binding affinity towards the target kinase as well as similar in vitro potency and ex vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. For example, 2-alkylimidazole 2, the analog of LN950, inhibits both the p38α MAP kinase as well as the LPS-stimulated tumor necrosis factor-α release from human whole blood in the low double-digit nanomolar range.

Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor.

Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors.

Scaffold modified Vemurafenib analogues as highly selective mitogen activated protein kinase kinase 4 (MKK4) inhibitors.

The mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified as druggable target for the treatment of acute liver failure in RNAi experiments. In these experiments MKK4 was identified to be a major regulator in hepatocyte regeneration. Inhibitors thereof may serve as medication to promote liver regeneration or reducing hepatocyte death. Just a small number of potent inhibitors with acceptable selectivity towards relevant off-targets are known up to date. Among the known potent inhibitors, selectivity is highly sensitive towards minor modifications of the molecule, which makes it necessary to carefully balance between potency and selectivity. In the herein presented study, a new class of Vemurafenib-derived inhibitors was investigated with α-carbolines as new scaffold. This new scaffold showed a remarkable intrinsic selectivity towards the chosen off-targets, without affecting potency towards MKK4 on a broad range of structural modifications.

Design and Synthesis of Highly Selective Brain Penetrant p38α Mitogen-Activated Protein Kinase Inhibitors.

Stress-induced p38α mitogen-activated protein (MAP) kinase activation modulates cytokine overproduction and is associated with neuroinflammation and neurodegeneration. As a potential therapeutic approach, novel Skepinone-based p38α MAP kinase inhibitors were optimized to cross the blood-brain barrier via either amino acid transporters or hydrophobic diffusion. To enhance absorption from the oral route, we used methyl ester prodrugs of the active carboxy analogs. Of these, 3-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (43; p38α, IC50 = 5.5 nM) and 4-(8-((2,4-difluorophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)butanoic acid (44; p38α, IC50 = 12 nM) had brain-to-plasma ratios of 1.4 and 4.4, respectively. Compound 70, 3-(8-((2-aminophenyl)amino)-5-oxo-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-carboxamido)propanoic acid (p38α, IC50 = 1.0 nM), the Skepinone-N counterpart of 43, was most present in the mouse brain (brain-to-plasma ratio of 4.7; 0.4 mg/kg p.o., 2 h, 580 nmol/kg). Compounds 43, 44, and 70 were p38α-MAP-kinase-selective, metabolically stable, hERG nonbinding, and able to modulate IL-6 and TNF-α production in cell-based assays.

Design and synthesis of 1H-pyrazolo[3,4-b]pyridines targeting mitogen-activated protein kinase kinase 4 (MKK4) - A promising target for liver regeneration.

Currently, the therapeutic options for treatment of liver failure are very limited. As mitogen-activated protein kinase kinase 4 (MKK4) has recently been identified by in vivo RNAi experiments to be a major regulator in hepatocyte regeneration, we pursued the development of a small molecule targeting this protein kinase. Starting from the approved BRAFV600E inhibitor vemurafenib (8), that showed a high off-target affinity to MKK4 in an initial screening, we followed a scaffold-hopping approach, changing the core heterocycle from 1H-pyrrolo[2,3-b]pyridine to 1H-pyrazolo[2,3-b]pyridine (10). Affinity to MKK4 could be conserved while the selectivity against off-target protein kinases was slightly improved. Further modifications led to 58 and 59 showing high affinity to MKK4 in the low nanomolar range and excellent selectivity profile from mandatory multiparameter-optimization for the essential anti-targets (MKK7, JNK1) and off-targets (BRAF, MAP4K5, ZAK) in the MKK4 pathway. Herein we report the first selective MKK4 inhibitors in this class.

From off-to on-target: New BRAF-inhibitor-template-derived compounds selectively targeting mitogen activated protein kinase kinase 4 (MKK4).

The mitogen-activated protein kinase (MAP) kinase 4 (MKK4) was found to be a major regulator of liver regeneration and could be a valuable drug target addressing liver related diseases by restoring its intrinsic regenerative capacity. We report on the synthesis and optimization of novel MKK4 inhibitors following a target-hopping strategy from the FDA-approved BRAFV600E inhibitor PLX4032 (8). Applying an iterative multi-parameter optimization process we carved out essential structural features yielding in compounds with a low nanomolar affinity for MKK4 and excellent selectivity profiles against the main off-targets MKK7 and JNK1, which, upon relevant inhibition, would totally abrogate the pro-regenerative effect of MKK4 inhibition, as well as against the off-targets MAP4K5, ZAK and BRAF with selectivity factors ranging from 40 to 430 for our best-balanced compounds 70 and 73.

Design and synthesis of novel fluorescently labeled analogs of vemurafenib targeting MKK4.

The mitogen-activated protein kinase kinase 4 (MKK4) plays a key role in liver regeneration and is under investigation as a target for stimulating hepatocytes to increased proliferation. Therefore, new small molecules inhibiting MKK4 may represent a promising approach for treating acute and chronic liver diseases. Fluorescently labeled compounds are useful tools for high-throughput screenings of large compound libraries. Here we utilized the azaindole-based scaffold of FDA-approved BRAF inhibitor vemurafenib 1, which displays off-target activity on MKK4, as a starting point in our fluorescent compound design. Chemical variation of the scaffold and optimization led to a selection of fluorescent 5-TAMRA derivatives which possess high binding affinities on MKK4. Compound 45 represents a suitable tool compound for Fluorescence polarization assays to identify new small-molecule inhibitors of MKK4.

Tri- and tetrasubstituted imidazoles as p38α mitogen-activated protein kinase inhibitors.

The synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles as potent p38α mitogen-activated protein kinase inhibitors is described. The trisubstituted imidazole series was found to be more potent than the tetrasubstituted imidazole series. Many of these compounds show low-nanomolar activities in the isolated p38α MAP kinase inhibition assay. The structure-activity relationships between these two series are different and not comparable.

2-(4-Fluoro-phen-yl)-N-{4-[6-(4-fluoro-phen-yl)-2,3-dihydro-imidazo[2,1-b][1,3]thia-zol-5-yl]pyridin-2-yl}acetamide.

In the crystal structure of the title compound, C(24)H(18)F(2)N(4)OS, the imidazole system makes dihedral angles of 34.3 (1) and 43.9 (1)°, respectively, with the directly attached 4-fluoro-phenyl and pyridine rings. The crystal structure is stabilized by inter-molecular N-H⋯N hydrogen bonding and by an intra-molecular C-H⋯O hydrogen inter-action. The F atom of the 2-(4-fluoro-phen-yl) group is disordered over two positions with site-occupancy factors of 0.75 and 0.25.

4-Chloro-7-methoxy-methyl-2-phenyl-7H-pyrrolo[2,3-b]pyridine.

In the title compound, C(15)H(13)ClN(2)O, the phenyl group makes a dihedral angle of 7.91 (8)° with the pyrrole ring. The crystal structure forms a three-dimensional network stabilized by π-π inter-actions [centroid-centroid distances = 3.807 (1) Å] between the pyridine and phenyl rings and via inter-molecular C-H⋯O hydrogen bonds.

2-(Bicyclo-[2.2.1]hept-5-en-2-yl)-1H-pyrrolo-[2,3-b]pyridine.

The crystal structure of the title compound, C(14)H(14)N(2), displays inter-molecular N-H⋯N hydrogen bonds, forming dimers of enanti-omeric mol-ecules via a crystallographic centre of inversion.

N-(4-Chloro-pyridin-2-yl)-N-(4-methyl-phenyl-sulfon-yl)acetamide.

The crystal structure of the title compound, C(14)H(13)ClN(2)O(3)S, features a three-dimensional network stabilized by inter-molecular C-H⋯O hydrogen bonds between the mol-ecules. The 4-methyl-phenyl-sulfonyl ring forms a dihedral angle of 30.6 (1)° with the 4-chloro-pyridine ring.

N-(4-Chloro-pyridin-2-yl)-N-meth-oxy-methyl-4-methyl-benzene-sulfonamide.

In the crystal structure of the title compound, C(14)H(15)ClN(2)O(3)S, each mol-ecule is connected via inter-molecular C-H⋯O hydrogen bonds to three further mol-ecules, generating a three-dimensional network. The 4-methyl-phenyl-sulfonyl ring forms a dihedral angle of 40.7 (2)° with the 4-chloro-pyridine ring.

7-[4-(4-Fluoro-phen-yl)-2-methyl-sulfanyl-1H-imidazol-5-yl]tetra-zolo[1,5-a]pyridine.

The crystal structure of the title compound, C(15)H(11)FN(6)S, forms a three-dimensional network stabilized by π-π inter-actions between the imidazole core and the tetra-zole ring of the tetra-zolopyridine-unit; the centroid-centroid distance is 3.627 (1) Å. The crystal structure also displays bifurcated N-H⋯(N,N) hydrogen bonding and C-H⋯F inter-actions. The former involve the NH H atom of the imidazole core and the tetra-zolopyridine N atoms, while the latter involve a methyl H atom, of the methyl-sulfanyl group, and the 4-fluoro-phenyl F atom. In the mol-ecule, the imidazole ring makes dihedral angles of 40.45 (9) and 17.09 (8)°, respectively, with the 4-fluoro-phenyl ring and the tetra-zolopyridine ring mean plane.

Influence of p38MAPK inhibition on IL-1beta-stimulated human chondrocytes: a microarray approach.

Articular chondrocytes respond to extracellular influences by activating signaling pathways which change gene expression. One key signal transduction pathway of inflammatory joint disease is mediated by the p38MAPK which is known to be activated by the pro-inflammatory cytokine IL-1beta. We used the p38MAPK inhibitor SB203580 and a whole human genome microarray in an in vitro inflammation model to identify genes regulated by this pathway in human chondrocytes. We found that 1,141 genes were regulated by IL-1beta, and 646 genes were regulated by the inhibitor whereas 116 genes were co-regulated by both substances. To elucidate the overall effect of SB203580, a GoMiner pathway analysis was performed which revealed involvement of versatile biological processes. Predominantly affected terms were 'response to stimulus', 'oxygen metabolism' and 'ligase activity'. We discuss herein the relevance and function of affected fields including the involved genes and unexpected effects of p38MAPK inhibition as it relates to the context of cartilage. Our results do not predict a pro-apoptotic or cancer promoting effect and markedly extend the knowledge on p38MAPK inhibition in chondrocytes beyond primary target genes.

4-(4-Fluoro-phen-yl)-1-methoxy-methyl-2-phenyl-1H-imidazole.

In the crystal structure of the title compound, C(17)H(15)FN(2)O, the mol-ecules form a three-dimensional network stabilized by π-π inter-actions between two imidazole rings related by a centre of symmetry. The distance between the centroids is 3.5488 (8) Å. The imidazole ring makes dihedral angles of 14.30 (7) and 33.39 (7)° with the 4-fluoro-phenyl ring and the phenyl ring, respectively.