Design,Synthesis, and Biological Evaluation of Tetrahydro‐β‐carboline Derivatives as Selective Sub‐Nanomolar Gelatinase Inhibitors

Targeting matrix metalloproteinases (MMPs) is a pursued strategy for treating several pathological conditions, such as multiple sclerosis and cancer. Herein, a series of novel tetrahydro‐β‐carboline derivatives with outstanding inhibitory activity toward MMPs are present. In particular, compounds 9 f , 9 g , 9 h and 9 i show sub‐nanomolar IC₅₀ values. Interestingly, compounds 9 g and 9 i also provide remarkable selectivity toward gelatinases; IC₅₀=0.15 nm for both toward MMP‐2 and IC₅₀=0.63 and 0.58 nm , respectively, toward MMP‐9. Molecular docking simulations, performed by employing quantum mechanics based partial charges, shed light on the rationale behind binding involving specific interactions with key residues of S1′ and S3′ domains. Taken together, these studies indicate that tetrahydro‐β‐carboline represents a promising scaffold for the design of novel inhibitors able to target MMPs and selectively bias gelatinases, over the desirable range of the pharmacokinetics spectrum.     

Probing the S1′ Site for the Identification of Non‐Zinc‐Binding MMP‐2 Inhibitors

Matrix metalloproteinases (MMPs) are zinc‐dependent enzymes involved in several pathological states. Among them, MMP‐2 is a relevant therapeutic target because of its role in cancer development and progression. Many MMP inhibitors (MMPIs) have been discovered over the last 30 years, and the majority of them contain a functional group that binds the zinc ion (zinc‐binding group; ZBG). Unfortunately, no MMPIs have reached the market yet, owing to toxic effects due to unselective interactions of the ZBG. The new generation of MMPIs that do not bind the zinc ion could overcome problems of selectivity and toxicity, but have so far been developed only for MMP‐8, ‐12, and ‐13. In this work, a virtual screening protocol was established by combining ligand‐ and structure‐based methods to identify non‐zinc‐binding MMP‐2 inhibitors using a new‐generation MMP‐8 inhibitor as a probe to find unexplored interactions in the MMP‐2 S1′ site. The screening allowed the identification of micromolar MMP‐2 inhibitors that putatively avoid binding the zinc ion, as demonstrated by docking calculations. The LIA model, built to correlate predicted and experimental binding energies of the identified non‐zinc‐binding MMP‐2 hits, underpins the reliability of the predicted docking poses.     

Synthesis and Biological Evaluation of 1‐Methyl‐1H‐indole–Pyrazoline Hybrids as Potential Tubulin Polymerization Inhibitors

A series of 1‐methyl‐1H‐indole–pyrazoline hybrids were designed, synthesized, and biologically evaluated as potential tubulin polymerization inhibitors. Among them, compound e19 [5‐(5‐bromo‐1‐methyl‐1H‐indol‐3‐yl)‐3‐(3,4,5‐trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide] showed the most potent inhibitory effect on tubulin assembly (IC₅₀=2.12 μm ) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC₅₀ values of 0.21–0.31 μm ). Further studies confirmed that compound e19 can induce HeLa cell apoptosis, cause cell‐cycle arrest in G₂/M phase, and disrupt the cellular microtubule network. These studies, along with molecular docking and 3D‐QSAR modeling, provide an important basis for further optimization of compound e19 as a potential anticancer agent.     

Synthesis and Biological Characterisation of Novel N‐Alkyl‐Deoxynojirimycin α‐Glucosidase Inhibitors

Illuminating glucosidases : The shown photoaffinity probe for endoplasmic reticulum (ER) α‐glucosidases was found to be a highly potent inhibitor of α‐glucosidase I in vitro and equally effective at inhibiting cellular ER glucosidases, as determined by a free oligosaccharide (FOS) analysis.

     

Fragment‐Based Discovery of 5‐Arylisatin‐Based Inhibitors of Matrix Metalloproteinases 2 and 13

Matrix metalloproteinases (MMPs) are well‐established targets for several pathologies. In particular, MMP‐2 and MMP‐13 play a prominent role in cancer progression. In this study, a structure‐based screening campaign was applied to prioritize metalloproteinase‐oriented fragments. This computational model was applied to a representative fragment set from the publically available EDASA Scientific compound library. These fragments were prioritized, and the top‐ranking hits were tested in a biological assay to validate the model. Two scaffolds showed consistent activity in the assay, and the isatin‐based compounds were the most interesting. These latter fragments have significant potential as tools for the design and realization of novel MMP inhibitors. In addition to their micromolar activity, the chemical synthesis affords flexible and creative access to their analogues.     

Bidentate Zinc Chelators for α‐Carbonic Anhydrases that Produce a Trigonal Bipyramidal Coordination Geometry

A series of new zinc binding groups (ZBGs) has been evaluated kinetically on 13 carbonic anhydrase (CA) isoforms. The fragments show affinity for all isoforms with IC₅₀ values in the range of 2–11 μM . The crystal structure of hCA II in complex with one such fragment reveals a bidentate binding mode with a trigonal‐bipyramidal coordination geometry at the Zn²⁺ center. The fragment also interacts with Thr199 and Thr200 through hydrogen bonding and participates in a water network. Further development of this ZBG should increase the binding affinity leading to a structurally distinct and promising class of CA inhibitors.     

Synthesis and Biological Testing of N‐Aminoimidazole‐Based p38α MAP Kinase Inhibitors

The p38 mitogen‐activated protein (MAP) kinase α plays a central role in the regulation of cellular responses such as differentiation, proliferation, apoptosis, and inflammation. Inhibition of p38 results in decreased synthesis of pro‐inflammatory cytokines. To date, diverse p38α inhibitors are in phase II clinical trials for numerous cytokine‐dependent diseases. 2‐Sulfanylimidazole derivatives offer advantages over the prototype inhibitor SB 203580, including fewer cytochrome P450 interactions and better kinetic properties. The aim of this study was to develop novel 1,2,4,5‐tetrasubstituted pyridinylimidazoles with acyl residues at the imidazole N1 position that can interact with the kinase's hydrophobic region II (HR II) or sugar pocket (SP) to improve both selectivity and activity. The substitution pattern was optimized by variation of the acyl moiety at the N1 position of the N‐aminoimidazole core. Acylation of the amino function was used for optimization and led to potent p38α MAPK inhibitors.     

Chemoenzymatic Synthesis of GDP‐L‐Fucose Derivatives as Potent and Selective α‐1,3‐Fucosyltransferase Inhibitors

Fucosyltransferases (FucTs) usually catalyze the final step of glycosylation and are critical to many biological processes. High levels of specific FucT activities are often associated with various cancers. Here we report the development of a chemoenzymatic method for synthesizing a library of guanosine diphosphate β‐L ‐fucose (GDP‐Fuc) derivatives, followed by in situ screening for inhibitory activity against bacterial and human α‐1,3‐FucTs. Several compounds incorporating appropriate hydrophobic moieties were identified from the initial screening. These were individually synthesized, purified and characterized in detail for their inhibition kinetics. Compound 5 had a K_i of 29 nM for human FucT‐VI, and is 269 and 11 times more selective than for Helicobacter pylori FucT (K_i=7.8 μM) and for human FucT‐V (K_i=0.31 μM).     

Organocatalytic Enantioselective Strecker Synthesis of α‐Quaternary α‐Trifluoromethyl Amino Acids

The first organocatalytic enantioselective Strecker synthesis of α‐quaternary α‐trifluoromethylated amino acids has been developed. Employing Takemoto’s thiourea catalyst the nucleophilic addition of trimethylsilyl cyanide to trifluoromethyl ketimines affords α‐amino nitriles in good to excellent yields (50–99%) and very good enantioselectivities (ee=83–95%). The enantiopure amino nitriles can be obtained by recrystallization. Deprotection and hydrolysis leads to the title amino acids.     

Immunoproteasome β5i‐Selective Dipeptidomimetic Inhibitors

N,C‐capped dipeptides belong to a class of noncovalent proteasome inhibitors. Herein we report that the insertion of a β‐amino acid into N,C‐capped dipeptides markedly decreases their inhibitory potency against human constitutive proteasome β5c, while maintaining potent inhibitory activity against human immunoproteasome β5i, thereby achieving thousands‐fold selectivity for β5i over β5c. Structure–activity relationship studies revealed that β5c does not tolerate the β‐amino acid based dipeptidomimetics as does β5i. In vitro, one such compound was found to inhibit human T cell proliferation. Compounds of this class may have potential as therapeutics for autoimmune and inflammatory diseases with less mechanism‐based cytotoxicity than agents that also inhibit the constitutive proteasome.     

Microbial Synthesis of Medium‐Chain α,ω‐Dicarboxylic Acids and ω‐Aminocarboxylic Acids from Renewable Long‐Chain Fatty Acids

Biotransformation of long‐chain fatty acids into medium‐chain α,ω‐dicarboxylic acids or ω‐aminocarboxylic acids could be achieved with biocatalysts. This study presents the production of α,ω‐dicarboxylic acids (e.g., C₉, C₁₁, C₁₂, C₁₃) and ω‐aminocarboxylic acids (e.g., C₁₁, C₁₂, C₁₃) directly from fatty acids (e.g., oleic acid, ricinoleic acid, lesquerolic acid) using recombinant Escherichia coli‐based biocatalysts. ω‐Hydroxycarboxylic acids, which were produced from oxidative cleavage of fatty acids via enzymatic reactions involving a fatty acid double bond hydratase, an alcohol dehydrogenase, a Baeyer–Villiger monooxygenase and an esterase, were then oxidized to α,ω‐dicarboxylic acids by alcohol dehydrogenase (ADH, AlkJ) from Pseudomonas putida GPo1 or converted into ω‐aminocarboxylic acids by a serial combination of ADH from P. putida GPo1 and an ω‐transaminase of Silicibacter pomeroyi. The double bonds present in the fatty acids such as ricinoleic acid and lesquerolic acid were reduced by E. coli‐native enzymes during the biotransformations. This study demonstrates that the industrially relevant building blocks (C₉ to C₁₃ saturated α,ω‐dicarboxylic acids and ω‐aminocarboxylic acids) can be produced from renewable fatty acids using biocatalysis.

     

Design,Synthesis, and Biological Evaluation of Levoglucosenone‐Derived Ras Activation Inhibitors

A panel of new potential Ras ligands was generated by decorating a tricyclic levoglucosenone‐derived scaffold with aromatic moieties. Some members of the panel show in vitro inhibitory activity toward the nucleotide exchange process on Ras and are toxic to some human cancer cell lines.

     

Design,Synthesis, and Biological Evaluation of Enantiomeric β‐N‐Acetylhexosaminidase Inhibitors LABNAc and DABNAc as Potential Agents against Tay‐Sachs and Sandhoff Disease

Combating glycolipid storage disorders : LABNAc was prepared in an efficient 11‐step procedure from D ‐lyxonolactone. The enantiomer DABNAc was also prepared from L ‐lyxonolactone. Preliminary cellular studies indicate that these compounds may find utility as chemical chaperones for the treatment of Tay‐Sachs and Sandhoff diseases.

     

3‐Heterocycle‐Phenyl N‐Alkylcarbamates as FAAH Inhibitors: Design,Synthesis and 3D‐QSAR Studies

Carbamates are a well‐established class of fatty acid amide hydrolase (FAAH) inhibitors. Here we describe the synthesis of meta‐substituted phenolic N‐alkyl/aryl carbamates and their in vitro FAAH inhibitory activities. The most potent compound, 3‐(oxazol‐2yl)phenyl cyclohexylcarbamate ( 2 a ), inhibited FAAH with a sub‐nanomolar IC₅₀ value (IC₅₀=0.74 nM ). Additionally, we developed and validated three‐dimensional quantitative structure–activity relationships (QSAR) models of FAAH inhibition combining the newly disclosed carbamates with our previously published inhibitors to give a total set of 99 compounds. Prior to 3D‐QSAR modeling, the degree of correlation between FAAH inhibition and in silico reactivity was also established. Both 3D‐QSAR methods used, CoMSIA and GRID/GOLPE, produced statistically significant models with coefficient of correlation for external prediction (R²_PRED) values of 0.732 and 0.760, respectively. These models could be of high value in further FAAH inhibitor design.     

Design,Synthesis, and Preliminary Biological Evaluation of Pyrrolo[3,4‐c]quinolin‐1‐one and Oxoisoindoline Derivatives as Aggrecanase Inhibitors

A small set of aggrecanase inhibitors based on the pyrrolo[3,4‐c]quinolin‐1‐one or oxoisoindoline frameworks bearing a 4‐(benzyloxy)phenyl substituent and different zinc binding groups were rationally designed and evaluated in silico by docking studies using the crystal structure of the ADAMTS‐5 catalytic domain (PDB code: 3B8Z). The designed compounds were synthesized via straightforward routes and tested for their potential inhibitory activity against ADAMTS‐5 and ADAMTS‐4. Among the compounds containing the pyrrolo[3,4‐c]quinolinone tricyclic system, hydroxamate derivative 2 b inhibited both ADAMTS‐5 and ADAMTS‐4, with IC₅₀ values in the submicromolar range and an inhibitory profile very similar to that of reference carboxylate derivative 11 . Conversely, the corresponding carboxylate derivative 2 a was significantly less active and unable to discriminate between ADAMTS‐5 and ‐4. The structure–activity relationship analysis of pyrroloquinolinone derivatives 2 a – i suggests that the carboxylate or hydroxamate groups of compounds 2 a , b play a key role in the interaction of these compounds with ADAMTS‐5 and ‐4. On the other hand, the oxoisoindoline derivatives 3 a , b lack significant ADAMTS‐4 inhibitory activity and inhibit ADAMTS‐5 showing IC₂₅ values in the micromolar range.     

Computer‐Guided Design,Synthesis, and Biological Evaluation of Quinoxalinebisarylureas as FLT3 Inhibitors

Activating mutations of FMS‐like tyrosine kinase 3 (FLT3) are present in ～30 % of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline‐based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced‐fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxalinebisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay.