Glycosaminoglycan Mimetics Obtained by Microwave-Assisted Sulfation of Marine Bacterium Sourced Infernan Exopolysaccharide.

Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell-cell and cell-matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.

Access and Modulation of Substituted Pyrrolo[3,4-c]pyrazole-4,6-(2H,5H)-diones.

The first access to polyfunctionnalized pyrrolo[3,4-c]pyrazole-4,6-(2H,5H)-dione derivatives is reported. The series were generated from diethyl acetylenedicarboxylate and arylhydrazines, which afforded the key intermediates bearing two functional positions. The annellation to generate the maleimide moiety of the bicycle was studied. Moreover, an efficient palladium-catalyzed C-C and C-N bond formation via Suzuki-Miyaura or Buchwald-Hartwig coupling reactions in C-6 position was investigated from 6-chloropyrrolo[3,4-c]pyrazole-4,6-(2H,5H)-diones. This method provides novel access to various 1,6 di-substituted pyrrolo[3,4-c] pyrazole-4,6-(2H,5H)-diones.

Selection of Bis-Indolyl Pyridines and Triphenylamines as New Inhibitors of SARS-CoV-2 Cellular Entry by Modulating the Spike Protein/ACE2 Interfaces.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC50) between 0.1 and 0.5 µM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.

Design and biological evaluation of substituted 5,7-dihydro-6H-indolo[2,3-c]quinolin-6-one as novel selective Haspin inhibitors.

A library of substituted indolo[2,3-c]quinolone-6-ones was developed as simplified Lamellarin isosters. Synthesis was achieved from indole after a four-step pathway sequence involving iodination, a Suzuki-Miyaura cross-coupling reaction, and a reduction/lactamization sequence. The inhibitory activity of the 22 novel derivatives was assessed on Haspin kinase. Two of them possessed an IC50 of 1 and 2 nM with selectivity towards a panel of 10 other kinases including the parent kinases DYRK1A and CLK1. The most selective compound exerted additionally a very interesting cell effect on the osteosarcoma U-2 OS cell line.

[18 F]-labeled positron emission tomography ligand for the histamine H4 receptor.

We synthesized 5-[18 F]-fluoro-1H-indol-2-yl)(4-methyl-1-piperazinyl)methanone ([18 F]5) via a Suzuki approach starting from a protected pinacol borane precursor followed by acidic hydrolysis of the t-Boc protecting group. The non-optimized radiochemical yield was 5.7 ± 1.35%, radiochemical purity was over 99%, and molar activity was 100.7 ± 34.5 GBq/µmol (n = 3). [18 F]5 was stable in rat plasma for at least 4 h and was evaluated by µPET imaging and biodistribution using a unilateral quinolinic acid rat model of neuroinflammation. The time-activity curve showed that [18 F]5 entered the brain immediately after intravenous injection and then left it progressively with a very low level reached from 30 min after injection. The biodistribution study showed no difference in the accumulation of [18 F]5 between the lesioned and intact side of the brain and between control rats and animals pretreated with a saturating dose of JNJ-7777120 as a specific H4R antagonist. Hence, despite its in vitro nanomolar affinity for H4R, and its ability to cross the blood-brain barrier in rats, [18 F]5 does not appear suitable to image in vivo the receptor by PET.

Design, Synthesis, In Silico and In Vitro Studies for New Nitric Oxide-Releasing Indomethacin Derivatives with 1,3,4-oxadiazole-2-thiol Scaffold.

Starting from indomethacin (IND), one of the most prescribed non-steroidal anti-inflammatory drugs (NSAIDs), new nitric oxide-releasing indomethacin derivatives with 1,3,4-oxadiazole-2-thiol scaffold (NO-IND-OXDs, 8a-p) have been developed as a safer and more efficient multitarget therapeutic strategy. The successful synthesis of designed compounds (intermediaries and finals) was proved by complete spectroscopic analyses. In order to study the in silico interaction of NO-IND-OXDs with cyclooxygenase isoenzymes, a molecular docking study, using AutoDock 4.2.6 software, was performed. Moreover, their biological characterization, based on in vitro assays, in terms of thermal denaturation of serum proteins, antioxidant effects and the NO releasing capacity, was also performed. Based on docking results, 8k, 8l and 8m proved to be the best interaction for the COX-2 (cyclooxygense-2) target site, with an improved docking score compared with celecoxib. Referring to the thermal denaturation of serum proteins and antioxidant effects, all the tested compounds were more active than IND and aspirin, used as references. In addition, the compounds 8c, 8h, 8i, 8m, 8n and 8o showed increased capacity to release NO, which means they are safer in terms of gastrointestinal side effects.

Design, Synthesis and SAR in 2,4,7-Trisubstituted Pyrido[3,2-d]Pyrimidine Series as Novel PI3K/mTOR Inhibitors.

This work describes the synthesis, enzymatic activities on PI3K and mTOR, in silico docking and cellular activities of various uncommon 2,4,7 trisubstituted pyrido[3,2-d]pyrimidines. The series synthesized offers a chemical diversity in C-7 whereas C-2 (3-hydroxyphenyl) and C-4 groups (morpholine) remain unchanged, in order to provide a better understanding of the molecular determinants of PI3K selectivity or dual activity on PI3K and mTOR. Some C-7 substituents were shown to improve the efficiency on kinases compared to the 2,4-di-substituted pyrimidopyrimidine derivatives used as references. Six novel derivatives possess IC50 values on PI3Kα between 3 and 10 nM. The compounds with the best efficiencies on PI3K and mTOR induced micromolar cytotoxicity on cancer cell lines possessing an overactivated PI3K pathway.

Design of new disubstituted imidazo[1,2-b]pyridazine derivatives as selective Haspin inhibitors. Synthesis, binding mode and anticancer biological evaluation.

Haspin is a mitotic protein kinase required for proper cell division by modulating Aurora B kinase localisation and activity as well as histone phosphorylation. Here a series of imidazopyridazines based on the CHR-6494 and Structure Activity Relationship was established. An assessment of the inhibitory activity of the lead structures on human Haspin and several other protein kinases is presented. The lead structure was rapidly optimised using a combination of crystal structures and effective docking models, with the best inhibitors exhibiting potent inhibitory activity on Haspin with IC50 between 6 and 100 nM in vitro. The developed inhibitors displayed anti-proliferative properties against various human cancer cell lines in 2D and spheroid cultures and significantly inhibited the migration ability of osteosarcoma U-2 OS cells. Notably, we show that our lead compounds are powerful Haspin inhibitors in human cells, and did not block G2/M cell cycle transition due to improved selectivity against CDK1/CyclinB.

Design of selective COX-2 inhibitors in the (aza)indazole series. Chemistry, in vitro studies, radiochemistry and evaluations in rats of a [18F] PET tracer.

A series of novel derivatives exhibiting high affinity and selectivity towards the COX-2 enzyme in the (aza) indazole series was developed. A short synthetic route involving a bromination/arylation sequence under microwave irradiation and direct C-H activation were established in the indazole and azaindazole series respectively. In vitro assays were conducted and structural modifications were carried out on these scaffolds to furnish compound 16 which exhibited effective COX-2 inhibitory activity, with IC50 values of 0.409 µM and an excellent selectivity versus COX-1. Radiolabeling of this most potent derivative [18F]16 was achieved after boron ester release and the tracer was evaluated in vivo in a rat model of neuroinflammation. All chemistry, radiochemistry and biological experimental data are discussed.

Regioselective Synthesis of New 2,4-(Het)aryl-3H-pyrido[1',2':1,5]pyrazolo[4,3-d]pyrimidines Involving Palladium-Catalyzed Cross-Coupling Reactions.

The design of some novel di-(het)arylated-3H-pyrido[1',2':1,5]pyrazolo[4,3-d]pyrimidine derivatives is reported. The series was developed from 1-aminopyridinium iodide, which afforded the key intermediate bearing two thiomethyl and amide functions, each of them useful for palladium catalyzed cross coupling reactions by alkyl sulfur release and C-O activation, respectively. The two regioselective and successive cross-coupling reactions were first carried out in C-4 by in situ C-O activation and next in C-2 by a methylsulfur release. Process optimization furnished conditions leading to products in high yields. The scope and limitations of the methodologies were evaluated and the final compounds characterized.

Synthesis of [1,3,4]Thiadiazolo[3',2':1,2]imidazo[4,5-c]quinolines including Pictet-Spengler Reaction and Exploration of Their C-2 Reactivity through SNAr.

This work reports the design of [1,3,4]thiadiazolo[3',2':1,2]imidazo[4,5-c]quinolines using a Pictet-Spengler reaction. The scope of the reaction was achieved from 6-(2-aminophenyl)imidazo[2,1-b][1,3,4]thiadiazole derivatives and available aldehydes. A wide range of aldehydes were employed to examine the scope of the cyclization. In parallel, a mechanism investigation was realized and showed a hydride transfer which led to a dismutation of the intermediate species. To complete this methodological study, a "sequential" oxidation/SNAr procedure was performed to achieve C-2 nucleophilic substitution using several amine types.

Controlled Dimroth Rearrangement in the Suzuki-Miyaura Cross Coupling of Triazolopyridopyrimidines.

Polynitrogen heterocycles are often subject to Dimroth rearrangement which consists of ring opening, bond rotation, and ring closure. In this note, we report a synthesis of two new families of triazolopyridopyrimidines. Successful functionalization via a Suzuki-Miyaura coupling was performed with total control of triazole (Dimroth) isomerization based on the judicious choice of reaction conditions.

New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway.

Human kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the α isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z'), and variability (r (2)). This CE method was easily extended to assay the inhibition of the ß, γ, and δ isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested.

The azaindole framework in the design of kinase inhibitors.

This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD) programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.

Design, synthesis and the biological evaluation of new 1,3-thiazolidine-4-ones based on the 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one scaffold.

New thiazolidine-4-one derivatives based on the 4-aminophenazone (4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) scaffold have been synthesized as potential anti-inflammatory drugs. The pyrazoline derivatives are known especially for their antipyretic, analgesic and anti-inflammatory effects, but recently there were synthesized new compounds with important antioxidant, antiproliferative, anticancer and antidiabetic activities. The beneficial effects of these compounds are explained by nonselective inhibition of cyclooxygenase izoenzymes, but also by their potential scavenging ability for reactive oxygen and nitrogen species. The structure of the new compounds was proved using spectroscopic methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the ferric reducing antioxidant power, phosphomolydenum reducing antioxidant power, DPPH and ABTS radical scavenging assays. The chemical modulation of 4-aminophenazone (6) through linkage to thiazolidine-propanoic acid derivatives 5a-l led to improved antioxidant potential, all derivatives 7a-l being more active than phenazone. The most active compounds are the derivatives 7e, and 7k, which showed the higher antioxidant effect depending on the antioxidant assay considered.

Synthesis and biological evaluation of new 1,3-thiazolidine-4-one derivatives of 2-(4-isobutylphenyl)propionic acid.

New thiazolidine-4-one derivatives of 2-(4-isobutylphenyl)propionic acid (ibuprofen) have been synthesized as potential anti-inflammatory drugs. The structure of the new compounds was proved using spectral methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the total antioxidant activity, the DPPH and ABTS radical scavenging assays. Reactive oxygen species (ROS) and free radicals are considered to be involved in many pathological events like diabetes mellitus, neurodegenerative diseases, cancer, infections and more recently, in inflammation. It is known that overproduction of free radicals may initiate and amplify the inflammatory process via upregulation of genes involved in the production of proinflammatory cytokines and adhesion molecules. The chemical modulation of acyl hydrazones of ibuprofen 3a-l through cyclization to the corresponding thiazolidine-4-ones 4a-n led to increased antioxidant potential, as all thiazolidine-4-ones were more active than their parent acyl hydrazones and also ibuprofen. The most active compounds are the thiazolidine-4-ones 4e, m, which showed the highest DPPH radical scavenging ability, their activity being comparable with vitamin E.

3-[(4-Phen-oxy-phen-yl)sulfan-yl]-5-phenyl-1H-1,2,4-triazole.

The title compound, C20H15N3OS, is V-shaped. In the 4-phen-oxy-phenyl group, the two rings are inclined to one another by 74.52 (13)°. These rings are inclined to the triazole ring by 72.20 (15) and 72.30 (15)°, respectively. The phenyl ring is inclined to the triazole ring by 10.85 (12)°. In the crystal, mol-ecules are linked via N-H⋯N hydrogen bonds, forming chains propagating along [010]. These chains are linked via pairs of C-H⋯S hydrogen bonds, forming sheets lying parallel to the ac plane.

Investigation of Ln3+ complexation by a DOTA derivative substituted by an imidazothiadiazole: synthesis, solution structure, luminescence and relaxation properties.

We investigated the coordination properties of original macrocyclic Ln3+ complexes comprising an imidazothiadiazole heterocycle. The thermodynamic stability of the Gd3+ complex was determined by a combination of potentiometric and photophysical measurements. The kinetic inertness was assessed in highly acidic media. The solution structure of the Ln3+ complex was unambiguously determined by a set of photophysical measurements and 1H, 13C, 89Y NMR data in combination with DFT calculations, which proved coordination of the heterocycle to Ln3+. The ability of the imidazothiadiazole moiety to sensitize Tb3+ luminescence was investigated. Finally, the relaxation properties were investigated by recording 1H nuclear magnetic relaxation dispersion (NMRD) profiles and 17O measurements. The water exchange rate is similar to that of GdDOTA as the less negative charge of the ligand is compensated for by the presence of a bulky heterocycle. Relaxivity is constant over a large range of pH values, demonstrating the favorable properties of the complex for imaging purposes.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Electrophoretically mediated microanalysis for in-capillary electrical cell lysis and fast enzyme quantification by capillary electrophoresis.

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. ß-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-ß-D-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the ß-galactosidase--released from the lysed cells--by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.