Synthesis of Polysubstituted Pyridines and Pyrazines via Truce-Smiles Rearrangement of Amino Acid-Based 4-Nitrobenzenesulfonamides.

Immobilized l-glutamic acid ß-methyl ester was sulfonylated with 4-nitrobenzenesulfonyl chloride and alkylated with various α-haloketones. The resulting sulfonamides were reacted with potassium trimethylsilanolate. Then, upon cleavage from the polymer support, tetrasubstituted pyridines were produced as the result of one-step C-arylation, aldol condensation, and oxidation. When cleavage from the resin occurred before the trimethylsilanolate treatment, C-arylation was followed by enamination, which yielded trisubstituted pyrazines. Through the developed protocols, targeted synthesis of novel heterocyclic derivatives was performed using mild reaction conditions and a number of readily available starting materials.

Synthesis of Polycyclic Tetrahydroisoquinolines and Tetrahydrobenzo[d]azepines from Polymer-Supported Allylglycine.

Herein, we report a multistep synthesis of polycyclic tetrahydroisoquinolines and tetrahydrobenzo[d]azepines starting from Wang resin-immobilized allylglycine. After sulfonylation with 2/4-nitrobenzenesulfonyl chlorides, Mitsunobu alkylation with various phenylalkynols yielded the corresponding (phenylprop-2-yn-1-yl)-sulfonamides. "Interior" ring-closure enyne metathesis (RCEM) using a Grubbs catalyst second generation (Ru2) yielded functionalized tetrahydroisoquinoline/tetrahydrobenzo[d]azepine intermediates. "East-side" [4 + 2] cycloaddition with representative dienophiles was followed by the "west-side" construction of different heterocycles using various electrophiles to finally furnish a set of novel molecular frameworks bearing fused [6 + 6] or [6 + 7] rings. The developed methodology enables the facile parallel synthesis of novel, pharmacologically promising compounds derived from privileged scaffolds.

Efficient synthesis of pentasubstituted pyrroles via intramolecular C-arylation.

Immobilized L-aspartic acid beta-methyl ester (Fmoc-Asp(OMe)-OH) was reacted with 4-nitrobenzenesulfonyl chloride, followed by alkylation with various α-haloketones. The resulting intermediates were treated with potassium trimethylsilanolate, which yielded tetrasubstituted pyrroles after a one-step transformation consisting of sequential C-arylation, aldol condensation and spontaneous aromatization. The discovered synthetic strategy enables fast and simple access to pentasubstituted and functionalized pyrroles from a number of readily available starting materials.

Reagent-Based Diversity-Oriented Synthesis of Triazolo[1,5-a][1,4]diazepine Derivatives from Polymer-Supported Homoazidoalanine.

Herein, we report the synthesis of skeletally different triazolo[1,5-a][1,4]diazepines starting from immobilized homoazidoalanine. After sulfonylation with 2/4-nitrobenzenesulfonyl chlorides and Mitsunobu alkylation with various alkynols, the corresponding N-substituted nitrobenzenesulfonamides were obtained. Their catalyst-free Huisgen cycloaddition provided immobilized and functionalized triazolo[1,5-a][1,4]diazepines as the key intermediates for further modification. Using the concept of diversity-oriented, reagent-based synthesis, the key intermediates were subsequently converted to heterocycles bearing [5 + 7 + 5], [5 + 7 + 6], and [5 + 7 + 7] scaffolds. Furthermore, the synthesis of spirocyclic triazolodiazepines was developed.

Cytoprotective activities of kinetin purine isosteres.

Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice.

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvß3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvß3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.

Rearrangement of Threonine- and Serine-Based N-(3-Phenylprop-2-yn-1-yl) Sulfonamides Yields Chiral Pyrrolidin-3-ones.

N-(3-Phenylprop-2-yn-1-yl)-sulfonamides derived from serine and threonine were synthesized using solid-phase synthesis and subjected to reaction with trimethylsilyl trifluoromethanesulfonate (TMSOTf). In contrast to the previously reported formation of 1,4-oxazepanes, this reaction afforded pyrrolidin-3-ones. A mechanistic explanation for this unexpected outcome is proposed, and the limitations and scope of the rearrangement are outlined.

A Synthetic Approach for the Rapid Preparation of BODIPY Conjugates and their use in Imaging of Cellular Drug Uptake and Distribution.

A solid-phase synthetic (SPS) method was developed for the preparation of BODIPY-labeled bioactive compounds that allows for fast and simple synthesis of conjugates for use in fluorescent microscopy. The approach was used to visualize cellular uptake and distribution of cytotoxic triterpenes in cancer cells.

Convenient Synthesis of Thiohydantoins, Imidazole-2-thiones and Imidazo[2,1-b]thiazol-4-iums from Polymer-Supported α-Acylamino Ketones.

The preparation of 5-methylene-thiohydantoins using solid-phase synthesis is reported in this paper. After sulfonylation of immobilized Ser (t-Bu)-OH with 4-nitrobenzenesulfonyl chloride followed by alkylation with various bromoketones, the 4-Nos group was removed and the resulting polymer-supported α-acylamino ketones reacted with Fmoc-isothiocyanate. Cleavage of the Fmoc protecting group was followed by the spontaneous cyclative cleavage releasing the 5-methylene-thiohydantoin derivatives from the polymer support. Reduction with triethylsilane (TES) yielded the corresponding 5-methyl-thiohydantoins. When Fmoc-isothiocyanate was replaced with alkyl isothiocyanates, the trifluoroacetic acid (TFA) mediated cleavage from the polymer support, which was followed by the cyclization reaction and the imidazo[2,1-b]thiazol-4-iums were obtained. Their conversion in deuterated dimethylsulfoxide led to imidazole-2-thiones.

A Stepwise Approach for the Synthesis of Folic Acid Conjugates with Protein Kinase Inhibitors.

Herein, we report an alternative synthetic approach for selected 2,6,9-trisubstituted purine CDK inhibitor conjugates with folic acid as a drug-delivery system targeting folate receptors. In contrast to the previously reported approaches, the desired conjugates were constructed stepwise using solid-phase synthesis starting from immobilized primary amines. The ability of the prepared conjugates to release the free drug was verified using dithiothreitol (DTT) and glutathione (GSH) as liberating agents. Finally, binding to the folate receptor (FOLR1) overexpressed in a cancer cell line was measured by flow cytometry using a fluorescent imaging probe.

Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin.

To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin-streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

N-acyl-ω-aminoaldehydes are efficient substrates of plant aminoaldehyde dehydrogenases.

Plant aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases and participate in the metabolism of compounds related to amino acids such as polyamines or osmoprotectants. Their broad specificity covers ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The substrate preference of plant AMADHs is determined by the presence of aspartic acid and aromatic residues in the substrate channel. In this work, 15 new N-acyl derivates of 3-aminopropanal (APAL) and 4-aminobutanal (ABAL) were synthesized and confirmed as substrates of two pea AMADH isoenzymes (PsAMADH 1 and 2). The compounds were designed considering the previously demonstrated conversion of N-acetyl derivatives as well as substrate channel dimensions (5-8 Å × 14 Å). The acyl chain length and its branching were found less significant for substrate properties than the length of the initial natural substrate. In general, APAL derivatives were found more efficient than the corresponding ABAL derivatives because of the prevailing higher conversion rates and lower K m values. Differences in enzymatic performance between the two isoenzymes corresponded in part to their preferences to APAL to ABAL. The higher PsAMADH2 affinity to substrates correlated with more frequent occurrence of an excess substrate inhibition. Molecular docking indicated the possible auxiliary role of Tyr163, Ser295 and Gln451 in binding of the new substrates. The only derivative carrying a free carboxyl group (N-adipoyl APAL) was surprisingly better substrate than ABAL in PsAMADH2 reaction indicating that also negatively charged aldehydes might be good substrates for ALDH10 family.

Plant ALDH10 family: identifying critical residues for substrate specificity and trapping a thiohemiacetal intermediate.

Plant ALDH10 family members are aminoaldehyde dehydrogenases (AMADHs), which oxidize ω-aminoaldehydes to the corresponding acids. They have been linked to polyamine catabolism, osmoprotection, secondary metabolism (fragrance), and carnitine biosynthesis. Plants commonly contain two AMADH isoenzymes. We previously studied the substrate specificity of two AMADH isoforms from peas (PsAMADHs). Here, two isoenzymes from tomato (Solanum lycopersicum), SlAMADHs, and three AMADHs from maize (Zea mays), ZmAMADHs, were kinetically investigated to obtain further clues to the catalytic mechanism and the substrate specificity. We also solved the high resolution crystal structures of SlAMADH1 and ZmAMADH1a because these enzymes stand out from the others regarding their activity. From the structural and kinetic analysis, we can state that five residues at positions 163, 288, 289, 444, and 454 (PsAMADHs numbering) can, directly or not, significantly modulate AMADH substrate specificity. In the SlAMADH1 structure, a PEG aldehyde derived from the precipitant forms a thiohemiacetal intermediate, never observed so far. Its absence in the SlAMADH1-E260A structure suggests that Glu-260 can activate the catalytic cysteine as a nucleophile. We show that the five AMADHs studied here are capable of oxidizing 3-dimethylsulfoniopropionaldehyde to the cryo- and osmoprotectant 3-dimethylsulfoniopropionate. For the first time, we also show that 3-acetamidopropionaldehyde, the third aminoaldehyde besides 3-aminopropionaldehyde and 4-aminobutyraldehyde, is generally oxidized by AMADHs, meaning that these enzymes are unique in metabolizing and detoxifying aldehyde products of polyamine degradation to nontoxic amino acids. Finally, gene expression profiles in maize indicate that AMADHs might be important for controlling ω-aminoaldehyde levels during early stages of the seed development.

Biological properties of pyrroloquinoline and pyrroloisoquinoline derivatives.

In this review, we summarize pyrroloquinoline and pyrroloisoquinoline derivatives (PQs and PIQs) that act on a broad spectrum of biological targets and are used as bacteriostatic, antiviral, plasmodial, anticancer, antidiabetic and anticoagulant agents. Many of these compounds play important roles in the study of DNA and its interactions, the regulation of the cell cycle and programmed cell death. This review involves twenty-five types of skeletally analogical compounds bearing pyrrole and (iso)quinoline scaffolds with different mutual annelations.

Switching on/off aryl hydrocarbon receptor and pregnane X receptor activities by chemically modified tryptamines.

Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.

Monoterpenoid aryl hydrocarbon receptor allosteric antagonists protect against ultraviolet skin damage in female mice.

The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1. As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR.

Plant aminoaldehyde dehydrogenases oxidize a wide range of nitrogenous heterocyclic aldehydes.

The metabolic degradation of aldehydes is catalyzed by oxidoreductases from which aldehyde dehydrogenases (EC 1.2.1) comprise nonspecific or substrate-specific enzymes. The latter subset is represented, e.g., by NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs; EC 1.2.1.19) oxidizing a group of naturally occurring ω-aminoaldehydes including polyamine oxidation products. Recombinant isoenzymes from pea (PsAMADH1 and 2) and tomato (LeAMADH1 and 2) were subjected to kinetic measurements with synthetic aldehydes containing a nitrogenous heterocycle such as pyridinecarbaldehydes and their halogenated derivatives, (pyridinylmethylamino)-aldehydes, pyridinyl propanals and aldehydes derived from purine, 7-deazapurine and pyrimidine to characterize their substrate specificity and significance of the resulting data for in vivo reactions. The enzymatic production of the corresponding carboxylic acids was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Although the studied AMADHs are largely homologous and supposed to have a very similar active site architecture, significant differences were observed. LeAMADH1 displayed the broadest specificity oxidizing almost all compounds followed by PsAMADH2 and 1. In contrast, LeAMADH2 accepted only a few compounds as substrates. Pyridinyl propanals were converted by all isoenzymes, usually better than pyridinecarbaldehydes and aldehydes with fused rings. The K (m) values for the best substrates were in the range of 10(-5)-10(-4) M. Nevertheless, the catalytic efficiency values (V (max)/K (m)) reached only a very small fraction of that with 3-aminopropanal (except for LeAMADH1 activity with two pyridine-derived compounds). Docking experiments using the crystal structure of PsAMADH2 were involved to discuss differences in results with position isomers or alkyl chain homologs.

Modulation of FLT3-ITD and CDK9 in acute myeloid leukaemia cells by novel proteolysis targeting chimera (PROTAC).

Oncogenic mutations in gene encoding FLT3 kinase are often detected in acute myeloid leukaemia (AML) patients, and several potent kinase inhibitors have been developed. However, the FLT3 inhibitor treatment often leads to the resistance development and subsequent relapse. Targeted degradation of oncogenic protein kinases has emerged as a feasible pharmacological strategy, providing more robust effect over traditional competitive inhibitors. Based on previously developed competitive inhibitor of FLT3 and CDK9, we have designed and prepared a novel pomalidomide-based PROTAC. A series of biochemical and cellular experiments showed selectivity towards FLT3-ITD bearing AML cells and confirmed proteasome-dependent mechanism of action. Dual FLT3-ITD and CDK9 protein degradation resulted in the block of FLT3-ITD downstream signalling pathways, apoptosis activation and cell cycle arrest of FLT3-ITD AML cells. Moreover, transcriptional repression caused by CDK9 degradation significantly reduced expression of crucial genes involved in AML pathogenesis. The obtained results indicate the beneficial impact of simultaneous FLT3-ITD/CDK9 degradation for AML therapy.

Purine scaffold effect on fluorescence properties of purine-hydroxyquinolinone bisheterocycles.

The fluorescence properties of bisheterocyclic compounds that contain purine and the 3-hydroxyquinolin-4(1H)-one skeleton connected with an aliphatic spacer of a different length/structure (3HQP) were examined. It was found that the introducing of the spacer-purine scaffold led in the comparison to 3HQs themselves to (1) the possibility of the effectual excitation in the wider range of excitation wavelengths, moreover, some derivatives can be excited at relatively high wavelengths around 400 nm, (2) the lowering of the quantum yield and (3) the slight longer wavelength shift of the dual emission spectra. Tested organic solvents did not affect significantly the 3HQP fluorescence properties. The characters of emission spectra as well as the quantum yields of 3HQPs were notably influenced by the ratio of water and DMSO in their composed mixture applied as a solvent. With increasing water content in the mixture both I(1)/I(2) and the quantum yield decreased.

1,4,6-Trisubstituted imidazo[4,5-c]pyridines as inhibitors of Bruton's tyrosine kinase.

Herein, we report an efficient synthetic approach towards trisubstituted imidazo [4,5-c]pyridines designed as inhibitors of Bruton's tyrosine kinase (BTK). Two alternative synthetic routes for the simple preparation of desired compounds with variable substitutions at the N1, C4, C6 positions were introduced with readily available building blocks. Further, the developed synthetic approach was feasible for isomeric compounds bearing imidazo [4,5-b]pyridine scaffolds. In contrast to expectations based on previous studies, the imidazo [4,5-c]pyridine inhibitor exhibited a significantly higher activity against BTK compared to its imidazo [4,5-b]pyridine isomer. An inherent SAR study in the series of imidazo [4,5-c]pyridine compounds revealed a remarkably high tolerance of C6 substitutions for both hydrophobic and hydrophilic substituents. Preliminary cellular experiments indicated selective BTK targeting in Burkitt lymphoma and mantle cell lymphoma cell lines. The inhibitors could thus serve as starting points for further development, eventually leading to BTK inhibitors that could be used after ibrutinib failure.