Mono- and bis(steroids) containing a cyclooctane core: Synthesis, antiproliferative activity, and action on cell cytoskeleton microtubules.

Steroid dimers of natural and synthetic origin possess an unusual and complex molecular architecture that may lead to the realization of peculiar effects in biological systems, in particular in different cancer cell lines. In the present work, diastereoselective ring-opening of mono- and polyoxiranes, containing a cyclooctane core, by azide-anion was performed to yield a series of azidoalcohols with different types of symmetry. The products were involved in copper-catalyzed azyde-alkyne cycloaddition (CuAAC) reaction with ethinylestradiol and ethinyltestosterone, and the resulting steroids and steroid dimers with triazole linkers were screened for their antiproliferative activity via (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. All the compounds revealed cytotoxicity toward several cancer cell lines. The effect of the most potent compound, containing two estradiol moieties, on the microtubules (MT) dynamics was investigated by immunofluorescent microscopy. The disruption of the majority of interphase cell cytoplasmic MT and mitotic event disturbances in the presence of the studied compound were observed. The latter effect caused the appearance of numerous multinucleated cells.

Verubulin (Azixa) Analogues with Increased Saturation: Synthesis, SAR and Encapsulation in Biocompatible Nanocontainers Based on Ca2+ or Mg2+ Cross-Linked Alginate.

Tubulin-targeting agents attract undiminished attention as promising compounds for the design of anti-cancer drugs. Verubulin is a potent tubulin polymerization inhibitor, binding to colchicine-binding sites. In the present work, a series of verubulin analogues containing a cyclohexane or cycloheptane ring 1,2-annulated with pyrimidine moiety and various substituents in positions 2 and 4 of pyrimidine were obtained and their cytotoxicity towards cancer and non-cancerous cell lines was estimated. The investigated compounds revealed activity against various cancer cell lines with IC50 down to 1-4 nM. According to fluorescent microscopy data, compounds that showed cytotoxicity in the MTT test disrupt the normal cytoskeleton of the cell in a pattern similar to that for combretastatin A-4. The hit compound (N-(4-methoxyphenyl)-N,2-dimethyl-5,6,7,8-tetrahydroquinazolin-4-amine) was encapsulated in biocompatible nanocontainers based on Ca2+ or Mg2+ cross-linked alginate and it was demonstrated that its cytotoxic activity was preserved after encapsulation.

3-[N,N-Bis(sulfonyl)amino]isoxazolines with Spiro-Annulated or 1,2-Annulated Cyclooctane Rings Inhibit Reproduction of Tick-Borne Encephalitis, Yellow Fever, and West Nile Viruses.

Spirocyclic compounds containing heterocyclic moieties represent promising 3D scaffolds for modern drug design. In the search for novel anti-flaviviral agents, we have obtained a series of 3-[N,N-bis(sulfonyl)amino]isoxazolines containing spiro-annulated cyclooctane rings and assessed their antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses. The structural analogs of spirocyclic compounds with a single sulfonyl group or 1,2-annulated cyclooctane ring were also investigated. Almost all the studied 3-[N,N-bis(sulfonyl)amino]isoxazolines revealed antiviral activity against TBEV and WNV. The most active against TBEV was spiro-isoxazoline derivative containing p-nitrophenyl groups in the sulfonyl part (EC50 2.0 ± 0.5 µM), while the highest potency against WNV was found for the compounds with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one (EC50 1.3 ± 0.5 µM). In summary, two novel scaffolds of anti-flaviviral agents based on N,N-bis(sulfonyl)amino]isoxazoline were proposed, and the compounds of this type demonstrated activity against TBEV and WNV.

Tetrahydroquinazoline N-oxide derivatives inhibit reproduction of tick-borne and mosquito-borne flaviviruses.

Tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and West Nile virus (WNV) are flaviviruses causing emerging arthropod-borne infections of a great public health concern. Clinically approved drugs are not available to complement or replace the existing vaccines, which do not provide sufficient coverage. Thus, the discovery and characterization of new antiflaviviral chemotypes would advance studies in this field. In this study, a series of tetrahydroquinazoline N-oxides was synthesized, and the antiviral activity of the compounds was assessed against TBEV, YFV, and WNV using the plaque reduction assay along with the cytotoxicity to the corresponding cell lines (porcine embryo kidney and Vero). Most of the studied compounds were active against TBEV (EC50 2 to 33 µM) and WNV (EC50 0.15 to 34 µM) and a few also demonstrated inhibitory activity against YFV (EC50 0.18 to 41 µM). To investigate the potential mechanism of action of the synthesized compounds, time-of-addition (TOA) experiments and virus yield reduction assays were performed for TBEV. The TOA studies suggested that the antiviral activity of the compounds should affect the early stages of the viral replication cycle after cell entry. Compounds with tetrahydroquinazoline N-oxide scaffold show a broad spectrum of activity against flaviviruses and represent a promising chemotype for antiviral drug discovery.

Novel Nanomolar Allosteric Modulators of AMPA Receptor of Bis(pyrimidine) Series: Synthesis, Biotesting and SAR Analysis.

Positive allosteric modulators (PAMs) of AMPA receptors represent attractive candidates for the development of drugs for the treatment of cognitive and neurodegenerative disorders. Dimeric molecules have been reported to have an especially potent modulating effect, due to the U-shaped form of the AMPA receptor's allosteric binding site. In the present work, novel bis(pyrimidines) were studied as AMPA receptor modulators. A convenient and flexible preparative approach to bis(pyrimidines) containing a hydroquinone linker was elaborated, and a series of derivatives with varied substituents was obtained. The compounds were examined in the patch clamp experiments for their influence on the kainate-induced currents, and 10 of them were found to have potentiating properties. The best potency was found for 2-methyl-4-(4-((2-methyl-5,6,7,8-tetrahydroquinazolin-4-yl)oxy)phenoxy)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidine, which potentiated the kainate-induced currents by up to 77% in all tested concentrations (10-12-10-6 M). The results were rationalized via the modeling of modulator complexes with the dimeric ligand binding domain of the GluA2 AMPA receptor, using molecular docking and molecular dynamics simulation. The prediction of ADMET, physicochemical, and PAINS properties of the studied bis(pyrimidines) confirmed that PAMs of this type may act as the potential lead compounds for the development of neuroprotective drugs.

Bis(oxiranes) Containing Cyclooctane Core: Synthesis and Reactivity towards NaN3.

Reactions of oxirane ring opening provide a powerful tool for regio- and stereoselective synthesis of polyfunctional and heterocyclic compounds, widely used in organic chemistry and drug design. Cyclooctane, alongside other medium-sized rings, is of interest as a novel molecular platform for the construction of target-oriented leads. Additionally, cyclooctane derivatives are well known to be prone to transannular reactions, which makes them a promising object in the search for novel approaches to polycyclic structures. In the present work, a series of cyclooctanediones was studied in Corey-Chaykovsky reactions, and novel spirocyclic bis(oxiranes) containing cyclooctane core, namely, 1,5-dioxadispiro[2.0.2.6]dodecane and 1,8-dioxadispiro[2.3.2.3]dodecane, were synthesized. Ring opening of the obtained bis(oxiranes) upon treatment with sodium azide was investigated, and it was found that the reaction path is determined by the reciprocal orientation of oxygen atoms in the oxirane moieties. Diastereomers of the bis(oxiranes) with cis-orientation underwent independent ring opening, supplying corresponding diazidodiols, while in the case of stereoisomers with trans-orientation, domino-like reactions occurred, including intramolecular nucleophilic attack and the formation of a novel three- or six-membered O-containing ring. Summarily, a straightforward approach to polyfunctional compounds containing cyclooctane or oxabicyclo[3.3.1]nonane cores, employing bis(oxiranes), was elaborated.

Bicyclic Isoxazoline Derivatives: Synthesis and Evaluation of Biological Activity.

The application of non-planar scaffolds in drug design allows for the enlargement of the chemical space, and for the construction of molecules that have more effective target-ligand interactions or are less prone to the development of resistance. Among the works of the last decade, a literature search revealed spirothiazamenthane, which has served as a lead in the development of derivatives active against resistant viral strains. In this work, we studied the novel molecular scaffold, which resembles spirothiazamenthane, but combines isoxazoline as a heterocycle and cyclooctane ring as a hydrophobic part of the structure. The synthesis of new 3-nitro- and 3-aminoisoxazolines containing spiro-fused or 1,2-annelated cyclooctane fragments was achieved by employing 1,3-dipolar cycloaddition of 3-nitro-4,5-dihydroisoxazol-4-ol 2-oxide or tetranitromethane-derived alkyl nitronates with non-activated alkenes. A series of spiro-sulfonamides was obtained by the reaction of 3-aminoisoxazoline containing a spiro-fused cyclooctane residue with sulfonyl chlorides. Preliminary screening of the compounds for antiviral, antibacterial, antifungal and antiproliferative properties in vitro revealed 1-oxa-2-azaspiro[4.7]dodec-2-en-3-amine and 3a,4,5,6,7,8,9,9a-octahydrocycloocta[d]isoxazol-3-amine with activity against the influenza A/Puerto Rico/8/34 (H1N1) virus in the submicromolar range, and high values of selectivity index. Further study of the mechanism of the antiviral action of these compounds, and the synthesis of their analogues, is likely to identify new agents against resistant viral strains.

A Facile Approach to Bis(isoxazoles), Promising Ligands of the AMPA Receptor.

A convenient synthetic approach to novel functionalized bis(isoxazoles), the promising bivalent ligands of the AMPA receptor, was elaborated. It was based on the heterocyclization reactions of readily available electrophilic alkenes with the tetranitromethane-triethylamine complex. The structural diversity of the synthesized compounds was demonstrated. In the electrophysiological experiments using the patch clamp technique on Purkinje neurons, the compound 1,4-phenylenedi(methylene)bis(5-aminoisoxazole-3-carboxylate) was shown to be highly potent positive modulator of the AMPA receptor, potentiating kainate-induced currents up to 70% at 10-11 M.

5-Nitroisoxazoles in SNAr reactions: access to polysubstituted isoxazole derivatives.

An efficient protocol for the straightforward functionalization of the isoxazole ring via the reactions of aromatic nucleophilic substitution of the nitro group with various nucleophiles has been elaborated. The method features excellent chemical yields, easy operability of the reaction, mild reaction conditions and a broad scope of both 5-nitroisoxazoles and nucleophiles. A synthetic approach to 3,5- and 3,4,5-substituted isoxazoles via the sequential functionalization of the isoxazole ring has been developed based on the excellent regioselectivity of the reaction of 3,5-dinitroisoxazoles with nucleophiles.

Spectrum of antiviral activity of 4-aminopyrimidine N-oxides against a broad panel of tick-borne encephalitis virus strains.

Tick-borne encephalitis is an important human arbovirus neuroinfection spread across the Northern Eurasia. Inhibitors of tick-borne encephalitis virus (TBEV) strain Absettarov, presumably targeting E protein n-octyl-ß-d-glucoside (ß-OG) pocket, were reported earlier. In this work, these inhibitors were tested in vitro against seven strains representing three main TBEV subtypes. The most potent compound, 2-[(2-methyl-1-oxido-5,6,7,8-tetrahydroquinazolin-4-yl)amino]-phenol, showed EC50 values lower than 22 µM against all the tested strains. Nevertheless, EC50 values for virus samples of certain strains demonstrated a substantial variation, which appeared to be consistent with the presence of E protein not only in infectious virions, but also in non-infectious and immature virus particles, protein aggregates, and membrane complexes.

Bivalent AMPA receptor positive allosteric modulators of the bis(pyrimidine) series.

The first example of a novel class of AMPA receptor positive allosteric modulators of the bis(pyrimidine) series having a hydroquinone linker has been obtained and showed a potency to increase kainate-induced currents at subnanomolar concentrations.

Unexpected Heterocyclization of Electrophilic Alkenes by Tetranitromethane in the Presence of Triethylamine. Synthesis of 5-Nitroisoxazoles.

A novel reaction of tetranitromethane with electrophilic alkenes in the presence of triethylamine affording substituted 5-nitroisoxazoles is described. Triethylamine reacts with tetranitromethane to generate N-nitrotriethylammonium and trinitromethanide. This process provides the heterocyclization of electrophilic alkenes. A variety of α,ß-unsaturated aldehydes, ketones, esters, amides, phosphonates, nitro, and sulfur compounds was involved in the heterocyclization reaction, and a wide range of functionalized 5-nitroisoxazoles was obtained in good to high yields. The scope and limitations of the reaction and the mechanistic proposal are discussed.

Substituent effects on stereoselectivity of dihalocarbene reactions with cyclohexadiene and on the reactivity of bis-dihalocyclopropanes in electrophilic nitrations en route to pyrimidine N-oxides.

Tricyclic bis-adducts of cyclohexa-1,4-diene with bromofluorocarbene and non-symmetric adducts with both bromofluoro- and dichlorocarbenes were synthesised selectively. The treatment of the bis-adducts with nitrating reagents in acetonitrile affords the products of heterocyclization of a sole dihalogenocyclopropane into 4-fluoropyrimidine N-oxide. The difference in the reactivity of bis-cyclopropanes with different sets of halogen substituents leads to selective heterocyclization of bromofluorocyclopropanes without affecting the dichlorocyclopropane moiety.

Beyond the Dimer and Trimer: Tetraspiro[2.1.2(5).1.2(9).1.2(13).1(3)] hexadecane-1,3,5,7-tetraone--the Cyclic Tetramer of Carbonylcyclopropane.

Tetraspiro[2.1.2(5).1.2(9).1.2(13).1(3)]hexadecane-1,3,5,7-tetraone 4, a unique tetraketone containing a cyclooctane core and four spiroannelated cyclopropane moieties, represents the previously unknown cyclotetramer of carbonylcyclopropane. For this purpose oxidation of the parent polyspirocyclic hydrocarbon was examined under various oxidative conditions, and the reactivity of oxidants towards methylene groups of the eight-membered cycle, activated by adjacent spirocyclopropane rings, was evaluated and contrasted. Whereas the treatment of tetraspirohexadecane with ozone resulted in monooxidation, its reaction with methyl(trifluoromethyl)dioxirane afforded the product of four-fold oxidation, triketoalcohol 10. Subsequent oxidation of the latter with Dess-Martin periodinane gave the target tetraketone 4.

Synthesis and assessment of 4-aminotetrahydroquinazoline derivatives as tick-borne encephalitis virus reproduction inhibitors.

Tick-borne encephalitis virus (TBEV) belonging to Flavivirus genus causes severe infection in humans. The search for therapeutically relevant compounds targeting TBEV requires the exploration of novel chemotypes. A versatile synthesis of previously unknown 4-aminopyrimidines and 4-aminopyrimidine N-oxides based on a fluorosubstituted heterocyclic core is described. A representative series of 4-aminotetrahydroquinazoline derivatives, containing aliphatic and aromatic substituents as well as the adamantane framework, was obtained and their activity against tick-borne encephalitis virus reproduction was studied. Nine compounds were found to inhibit TBEV entry into the host cells. A bulky hydrophobic adamantyl group was identified to be important for the antiviral activity. The developed synthetic route allowed an easy access to a consistent compound library for further structure-activity relationship studies.

symm-Tetramethylenecyclooctane: en route to polyspirocycles.

A straightforward gram-scale synthesis of 1,3,5,7-tetrakis(methylidene)cyclooctane (TMCO) from commercial adamantane-1,3-dicarboxylic acid has been developed. TMCO exhibits high reactivity toward a number of carbenes and epoxidizing reagents, undergoing multiple cyclopropanations, dihalocyclopropanations, or epoxidations of four double bonds to yield polyspirocyclic products. Stereochemical features of polyspirocyclopropanated compounds have been thoroughly examined in experimental (NMR) and theoretical (DFT) studies. Comprehensive stereochemical assignment of TMCO adducts with dihalocarbenes and spiroepoxy products was achieved. The conditions of the formation of 1-methyl-3,7-bis(methylidene)bicyclo[3.3.1]nonane from the adamantane derivative were optimized, and diadducts of this diene with dihalocarbenes were isolated and characterized.

Three-component heterocyclization of gem-bromofluorocyclopropanes with NOBF4: access to 4-fluoropyrimidine N-oxides.

Novel three-component heterocyclization involving gem-bromofluorocyclopropanes, nitrosyl tetrafluoroborate, and a molecule of the solvent (nitrile) yielding previously unknown fluorinated pyrimidine N-oxides is described. A two-step synthetic approach to 4-fluoropyrimidine N-oxides from alkenes under mild conditions is developed using this reaction. Mechanistic aspects of the heterocyclization are discussed.