Synthesis of 17α-substituted ethynylestradiols: Potential ligands for drug vectors

17α-substituted ethynylestradiols, derived from estrone, were converted to their corresponding 17α-(bromo- or iodo-propargyl)estrone intermediates. Nucleophilic substitution onto these moieties with malonate diester followed by hydrolysis and complexation with cis-Pt(Me₂en)I₂ (Me₂en = N,N-dimethylethylenediamine) gave cis-Pt(Me₂en)(2-(3-(17β-estradiol-17α-yl)-prop-2-ynyl)malonato) 7, thus demonstrating that these estrogen-derived compounds can be used to synthesize stable Pt(II) complexes. The 3-(17β-estradiol-17α-yl)-prop-2-ynyl-1-sulfanylethylthiol 23 was also prepared.     

Synthesis and biological evaluation of novel N,N′-diaryl cyanoguanidines acting as potent and selective carbonic anhydrase II inhibitors

A series of novel N,N′′-diaryl cyanoguanidines were synthesized by reacting diphenyl N-cyanocarbonimidate with sulfanilamide followed by treatment of the obtained cyano-O-phenylisourea with substituted aromatic amines. The newly prepared N,N′′-diaryl cyanoguanidines showed a very interesting inhibition profile against four selected human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, hCA I and hCA II (cytosolic), hCA IV (membrane-bound), and hCA IX (transmembrane). All these compounds showed a potent inhibition against isoform hCA II,with inhibition constants in the low nanomolar range, as well as a high selectivity for hCA II over hCA I, IV and IX. Since hCA II is an important drug target for antiglaucoma agents, these isoform-selective inhibitors may be considered of interest for further medicinal/pharmacologic studies.     

Synthesis of 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazole derivatives: Their evaluation as potential PDE 4B inhibitors possessing cytotoxic properties against cancer cells

The 2,2,4-trimethyl-1,2-dihydroquinolinyl substituted 1,2,3-triazole derivatives were designed as potential inhibitors of PDE4B. These compounds were synthesized via a multi-step sequence consisting of copper-catalyzed azide-alkyne cycloaddition (CuAAC) as a key step in aqueous media. The required alkynes were prepared from nimesulide via N-propargylation and then nitro group reduction followed by a CAN mediated modified Skraup reaction of the resulting amine. All the synthesized compounds showed PDE4B inhibitory properties in vitro at 30 μM with two compounds showing >50% inhibition that were supported by the in silico docking results of these compounds at the active site of PDE4B. Three of these PDE4 inhibitors showed promising cytotoxic properties against A549 human lung cancer cells in vitro with IC₅₀ ∼8–9 μM.     

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.     

Reactions of chitosan: 4. Preparation of organosoluble derivatives of chitosan

Fully subtituted di-O-acetyl-N-acetylchitosan (chitin diacetate) has been prepared by a route in which the hydroxyl groups are acetylated prior to N-acetylation. This overcomes the previously reported intramolecular steric hindrance to esterification caused by the N-acetamido group. The resultant products were of high viscosity but had a limited solubility range. Di-O-acrylcarbamate derivatives of N-acetylchitosan (chitin) have been produced by a similar route, whilst di-O-arylcarbamate-N-arylureidochitosans have been prepared directly from chitosan. These products also have limited solubility ranges and have inherent viscosities similar to that of di-O-acetylchitosan prepared from the same batch of chitosan.     

N-methyl-2-anilino-6-naphthalenesulfonyl hydrazine (2,6-mansyl hydrazine): A new sensitive fluorometric reagent for carbonyl compounds

An almost quantitative synthesis of N-methyl-2-anilino-6-naphthalenesulfonyl hydrazine (2,6-mansyl hydrazine) from sulfonyl chloride and hydrazinlum hydroxide is described. The 2,6-mansyl chloride was prepared by different methods from 6-hydroxynaphthalene-2-sulfonic acid (overall yield: 69%). The N- and the O-mansylation of suitable compounds (e.g., amines, amino acids, and phenolic steroids) with 2,6-mansyl chloride and the preparation of oxosteroid-2,6-mansyl hydrazones are deseribed, and the derivatives obtained, their uv spectra, and methods for their thin-layer chromatographic separation are compared with the corresponding data for dansylated compounds.     

Synthesis of 3'-azido-3'-deoxythymidine (AZT)--Cinchona alkaloid conjugates via click chemistry: Toward novel fluorescent markers and cytostatic agents

Novel nucleoside-Cinchona alkaloid conjugates were synthesized using ‘click’ chemistry approach based on the copper(I) catalyzed Huisgen azide-alkyne cycloaddition. Two series of conjugates were prepared employing 3′-azido-3′-deoxythymidine (AZT) as the azide component and the four 10,11-didehydro Cinchona alkaloids as well as their 9-O-propargyl ethers as the alkyne components. All obtained conjugates showed strong fluorescence emission and some of them exhibited marked cytotoxic activity in vitro.     

N-acetoxy-N-acetylaminoarenes and nitrosoarenes one-electron non-enzymatic and enzymatic oxidation products of various carcinogenic aromatic acethydroxamic acids

A number of carcinogenic aromatic acethydroxamic acids (e.g.N-hydroxy-N-acetyl derivatives of 2-aminofluorene, 3-aminofluorene, 4-aminostilbene, 1-aminonaphthalene, 2-aminonaphthalene, 2-aminophenanthrene, and 4-aminobiphenyl) are readily oxidized by alkaline Fe(CN)₆³⁻ or Ag₂O. The free nitroxide radicals thus formed dismutate in organic solution according to second order kinetics to yield the corresponding N-acetoxy-N-acetylaminoarenes and nitrosoarenes. The structures of the latter products were established by mass and infrared spectrum analyses. Evicence was obtained for a similar one-electron oxidation of these acethydroxamic acids with horseradish peroxidase and H₂O₂ at pH 7. One-electron oxidation of N-hydroxy-2-acetylaminofluorene was also demonstrated with lactoperoxidase and human myeloperoxidase. The possible relevance of a similar peroxidative attack in vivo to the carcinogenic activities of some aromatic amines and amides is discussed.     

Synthesis and characterization of a triazine dendrimer that sequesters iron(III) using 12 desferrioxamine B groups

The synthesis of a third generation triazine dendrimer, 1, containing multiple, iron-sequestering desferrioxamine B (DFO) groups is described. Benzoylation of the hydroxamic acid groups of DFO and formation of a reactive dichlorotriazine provide the intermediate for reaction with the second generation dendrimer displaying twelve amines. This strategy further generalizes the ‘functional monomer’ approach to generate biologically active triazine dendrimers. Dendrimer 1 is prepared in seven steps in 35% overall yield and displays 12 DFO groups making it 56% drug by weight. Spectrophotometric titrations (UV–vis) show that 1 sequesters iron(III) atoms with neither cooperativity nor significant interference from the dendrimer backbone. Evidence from NMR spectroscopy and mass spectrometry reveals a limitation to this functional monomer approach: trace amounts of O-to-N acyl migration from the protected hydroxamic acids to the amine-terminated dendrimer occurs during the coupling step leading to N-benzoylated dendrimers displaying fewer than 12 DFO groups.     

Synthesis and characterization of new inhibitors of cholinesterases based on N-phenylcarbamates: In vitro study of inhibitory effect,type of inhibition,lipophilicity and molecular docking

Based on current treatment of Alzheimer's disease, where the carbamate inhibitor Rivastigmine is used, two series of carbamate derivatives were prepared: (i) N-phenylcarbamates with additional carbamate group (1–12) and (ii) N-phenylcarbamates with monosaccharide moiety (13–24). All compounds were tested for the inhibitory effect on both of the cholinesterases, electric eel acetylcholinesterase (eeAChE) and butyrylcholinesterase from equine serum (eqBChE) and the inhibitory activity (expressed as IC₅₀ values) was compared with that of the established drugs Galanthamine and Rivastigmine. The compounds with two carbamate groups 1–12 revealed higher inhibitory efficiency on both cholinesterases in compared with monosaccharide derived carbamates 13–24 and with Rivastigmine. The significant decrease of inhibitory efficiency on eqBChE (also for eeAChE but in less manner) was observed after deacetalization of monosaccharide. Moreover, the type of inhibitory mechanism of five chosen compounds was studied. It was found, that compounds with two carbamate groups act presumably via a mixed inhibitory mechanism and the compounds with monosaccharide moiety act as non-competitive inhibitors. The lipophilicity of tested compounds was determined using partition coefficient. Specific positions of the inhibitors in the binding sites of cholinesterases were determined using molecular modeling and the results indicate the importance of phenylcarbamate orientation in the catalytic gorges of both enzymes.     

The use of positively charged leaving-groups in the synthesis of α-D-linked glucosides. Synthesis of methyl 2,3,4-tri-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside

Quaternary ammonium and triphenylphosphonium salts of 2,3,4-tri-O-benzyl-6-O-(N-phenylcarbamoyl)-D-glucopyranosyl bromide were readily prepared by reaction with tertiary amines and triphenylphosphine under anhydrous conditions. Methanolysis of these salts was studied to determine the conditions of solvent and temperature that would produce the highest yields of α-D-glucosides. The quaternary ammonium salts gave the highest yields with solvents of low dielectric constant and room temperature. The phosphonium salts gave moderate yields with diethyl ether at 50°. The synthesis of methyl 2,3,4-tri-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl)-α-D-glucopyranoside by treatment of the quaternary ammonium salt of 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl bromide with methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside was studied as a model for the synthesis of oligosaccharides. The anomeric composition of the disaccharide product could be easily determined from the optical rotation since the specific rotations of both the final product and of the gentiobioside analog are known. Under the best conditions, the yield of disaccharide was low (50%) and the reactions were not completely stereoselective.     

Flexible double-headed cytosine-linked 2′-deoxycytidine nucleotides. Synthesis,polymerase incorporation to DNA and interaction with DNA methyltransferases

New types of double-headed 2′-deoxycytidine 5′-O-triphosphates (dC^XCTPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC^XCTPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins.     

Synthesis of xylan-graft-poly(l-lactide) copolymers via click chemistry and their thermal properties

Di-O-(6-azidohexanoyl)-xylan-graft-poly(l-lactide)s (XylC6N₃-g-PLLAs) were prepared by grafting propargyl-terminated poly(l-lactide) onto di-O-(6-azidohexanoyl)-xylan (XylC6N₃) via click chemistry. Di-O-(6-azidohexanoyl)-xylan (XylC6N₃) was prepared via two steps from xylan extracted from eucalyptus kraft pulp with aqueous sodium hydroxide solution. Propargyl-terminated poly(l-lactide)s (PLLA) with three different molecular weights were synthesized via ring-opening polymerization of l-lactide using propargyl alcohol as initiator and tin (II) octanoate (Sn(Oct)₂) as catalyst. XylC6N₃ and propargyl-terminated PLLAs were treated with N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) and copper(I) bromide, and the graft copolymers XylC6N₃-g-PLLAs were obtained. DSC measurements revealed that the glass transition temperatures (T_g) of the copolymers decreased compared to that of XylC6N₃, suggesting that the grafted PLLA side-chains act as an internal plasticizer for xylan. TGA measurements revealed that XylC6N₃-g-PLLAs had higher decomposition temperatures than those of XylC6N₃ or PLLA, and that the decomposition temperatures of the copolymers increased with decrease in the number of PLLA side-chains grafted to the xylan main-chain.     

Synthesis and biological evaluation of novel propargyl amines as potential fluorine-18 labeled radioligands for detection of MAO-B activity

The aim of this project was to synthesize and evaluate three novel fluorine-18 labeled derivatives of propargyl amine as potential PET radioligands to visualize monoamine oxidase B (MAO-B) activity.The three fluorinated derivatives of propargyl amine ((S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)-pent-4-en-2-amine (5), (S)-N-(1-fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (10) and (S)-1-fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (15)) were synthesized in multi-step organic syntheses. IC₅₀ values for inhibition were determined for compounds 5, 10 and 15 in order to determine their specificity for binding to MAO-B. Compound 5 inhibited MAO-B with an IC₅₀ of 664 ± 48.08 nM. No further investigation was carried out with this compound. Compound 10 inhibited MAO-B with an IC₅₀ of 208.5 ± 13.44 nM and compound 15 featured an IC₅₀ of 131.5 ± 0.71 nM for its MAO-B inhibitory activity. None of the compounds inhibited MAO-A activity (IC₅₀ > 2 μM).The fluorine-18 labeled analogues of the two higher binding affinity compounds (10 and 15) (S)-N-(1-[¹⁸F]fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (16) and (S)-1-[¹⁸F]fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (18) were both prepared from the corresponding precursors 9A, 9B and 14A, 14B by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography experiments on human postmortem brain tissue sections were performed with 16 and 18. Only compound 18 demonstrated a high selectivity for MAO-B over MAO-A and was, therefore, chosen for further examination by PET in a cynomolgus monkey.The initial uptake of 18 in the monkey brain was 250% SUV at 4 min post injection. The highest uptake of radioactivity was observed in the striatum and thalamus, regions with high MAO-B activity, whereas lower levels of radioactivity were detected in the cortex and cerebellum. The percentage of unchanged radioligand 18 was 30% in plasma at 90 min post injection.In conclusion, compound 18 is a selective inhibitor of MAO-B in vitro and demonstrated a MAO-B specific binding pattern in vivo by PET in monkey. It can, therefore, be considered as a candidate for further investigation in human by PET.     

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.^1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.^4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.^1,2The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically¹ or analytically,^2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,^1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.^1,2,7In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N₃) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.     

Synthesis of new β-amidodehydroaminobutyric acid derivatives and of new tyrosine derivatives using copper catalyzed C–N and C–O coupling reactions

Several β-amidodehydroaminobutyric acid derivatives were prepared from N,C-diprotected β-bromodehydroaminobutyric acids and amides by a copper catalyzed C–N coupling reaction. The best reaction conditions include the use of a catalytic amount of CuI, N,N′-dimethylethylenediamine as ligand and K₂CO₃ as base in toluene at 110 °C. The stereochemistry of the products was determined using NOE difference experiments and the results obtained are in agreement with an E-stereochemistry. Thus, the stereochemistry is maintained in the case of the E-isomers of β-bromodehydroaminobutyric acid derivatives, but when the Z-isomers were used as substrates the reaction proceeds with inversion of configuration. The use of β-bromodehydrodipeptides as substrates was also tested. It was found that the reaction outcome depend on the stereochemistry of the β-bromodehydrodipeptide and on the nature of the first amino acid residue. The products isolated were the β-amidodehydrodipeptide derivatives and/or the corresponding dihydropyrazines. The same catalytic system (CuI/N,N′-dimethylethylene diamine) was used in the C–O coupling reactions between a tyrosine derivative and aryl bromides. The new O-aryltyrosine derivatives were isolated in moderate to good yields. The photophysical properties of two of these compounds were studied in four solvents of different polarity. The results show that these compounds after deprotection can be used as fluorescence markers.     

Synthesis of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)-2,3-diiodo-1-propene and its reaction with primary amines

Iodination of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)allene affords an E/Z-mixture of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)-2,3-diiodo-1-propene. S_N2 displacement of the allylic iodide with primary amines affords an E/Z-mixture of allylic amines under a variety of conditions. Due to its experimental simplicity and low cost of reagents, this procedure may find wide use in the laboratory for functionalization of sugar allenes.     

Stimulation of Methanogenesis by Aldicarb and Several Other N-Methyl Carbamate Pesticides

Aldicarb and several other N-methyl carbamate pesticides stimulated methane production in anaerobic salt marsh soils and organic-rich aquifer soils. Stimulation was biological and linearly related to the amount of carbamate added. Of the four carbamates studied, methomyl gave the greatest stimulation followed by carbaryl, aldicarb, and baygon. The percent conversions [(moles of CH₄ in excess of control/mole of carbamate added) × 100] for methomyl, carbaryl, aldicarb, and baygon were 88, 57, 40, and 11, respectively. Using aldicarb as a model carbamate, we found that monomethylamine (MA) accumulated in sediments as a result of aldicarb addition. MA arises from the N-methyl carbamoyl portion of the carbamates as a result of presumptive biological hydrolysis. MA levels decreased as CH₄ production was stimulated, and 2-bromoethane sulfonic acid (a specific inhibitor of mathanogenesis) partially inhibited the loss of MA. These findings suggest that N-methyl carbamates are readily hydrolyzed to MA in the presence of an active microbial population under anaerobic conditions and that methanogenesis is stimulated as a result of the consumption of MA by methanogenic bacteria.     

Compost amendments enhance peat suppressiveness to Pythium ultimum,Rhizoctonia solani and Sclerotinia minor

Peat is the most common organic material used for the preparation of potting mix because of its homogeneous and favorable agronomic characteristics. However, this organic material is poorly suppressive against soilborne pathogens and fungicides are routinely used to manage damping-off diseases. In the present study, we investigated the suppressive capability of five compost – peat mixtures towards the plant pathogens Pythium ultimum, Rhizoctonia solani and Sclerotinia minor – Lepidium sativum pathosystems. For all organic media, 18 parameters were measured including enzymatic activities (glucanase, N-acetyl-glucosaminidase, chitobiosidase and hydrolysis of fluorescein diacetate), microbiological (BIOLOG® EcoPlates?, culturable bacteria and fungi), and chemical features (pH, EC, total, extractable and humic carbon, total and organic N, NH₄–N, total protein and water content). In addition, ¹³C-CPMAS-NMR spectroscopy was used to characterize the organic materials. Peat amended with composts reduced disease damping-off caused by P. ultimum, R. solani and S. minor in 60% of the mixtures and compost derived from animal manure showed the largest and most consistent disease suppression. Sterilization decreased or eliminated suppressiveness of 42.8% of the mixtures. The most useful parameters to predict disease suppression were different for each pathogen: extractable carbon, O-aryl C and C/N ratio for P. ultimum, alkyl/O-alkyl ratio, N-acetyl-glucosaminidase and chitobiosidase enzymatic activities for R. solani and EC for S. minor. Our results demonstrate that the addition of composts to peat could be useful for the control of soilborne pathogens.     

Synthesis of coordination compounds via dehalogenation of zinc bromoacetate in presence of some amines

The dehalogenation reactions of pure zinc bromoacetate and its mixtures with hexamethylenetetramine, 2,2′-bipyridine, 1,10-phenanthroline were studied in water solutions. Due to the decomposition of hexamethylenetetramine during reaction, the cadmium bromoacetate-hexamethylenetetramine system was also studied. The four new coordination compounds, catena-[bis(μ₂-α-hydroxyacetato-κ³O¹,O²:O^1′)-zinc], catena[μ₂-1-(8-carboxylateethyl)-1,3,5,7-tetra-aza-adamantan-1-ium-N,O′]-dibromo-cadmium, dibromo-(2,2′-bipyridine-N,N′)-zinc, dibromo-(1,10-phenanthroline-N,N′)-zinc were synthesised and characterised by elemental and thermal analysis, IR and UV-vis spectroscopy, and X-ray crystallography. All compounds are air stable and well soluble in water. The zinc hydroxyacetate creates two-dimensional 4-c uninodal net. The zinc atoms are four and six coordinated and the cadmium atom is five coordinated. The coordination polyhedra of central atoms can be described as octahedron and trapezoidal pyramid for Zn and Cd polymers, respectively, and as trigonal pyramid for ZnBr₂ complexes. The bond valences considerations show that the chelating amines are bonded almost two times stronger to the central atoms than the chelating carboxylate groups. In the structures of polymeric compounds exist O-H?O, C-H?O, C-H?N and C-H?Br hydrogen bonds. The IR spectra show typical vibrations for chelating amine molecules. The thermal decomposition of studied compounds proceeds via multiple steps with gradual evolution of ligands.