Novel N‐Cycloalkylcarbonyl‐N′‐arylthioureas: Synthesis,Design, Antifungal Activity and Gene Toxicity

A synthesis method of novel N‐cycloalkylcarbonyl‐N′‐arylthioureas was developed. It consists of sequential addition of equimolecular amounts of ammonium isothiocyanate and substituted anilines to cycloalkylcarbonyl chlorides. The identity and purity of products were confirmed by LC/MS spectra, their structure by elemental analysis, IR and ¹H‐NMR spectra. Preliminary antimicrobial screening for standard microorganisms and molecular docking allowed to select several structures for antifungal and genetic toxicity studies. Conducted in vitro screening of 9 compounds for antifungal potential against 11 phytopathogenic fungi and three Phytophthora strains revealed that two N‐(arylcarbamothioyl) cyclopropanecarboxamides at a concentration of 50 μg/ml exhibited activities comparable to the standard antifungal agent ‘Cyproconazole’. Analysis of mutagenicity of novel thioureas using the Salmonella reverse mutagenicity assay (‘Ames Test’) showed a low gene‐toxicity profile.     

Synthesis of rigid tryptophan mimetics by the diastereoselective Pictet–Spengler reaction of β3‐homo‐tryptophan derivatives with chiral α‐amino aldehydes

The Pictet–Spengler (PS) cyclizations of β³‐hTrp derivatives as arylethylamine substrates were performed with L‐α‐amino and D‐α‐amino aldehydes as carbonyl components. During the PS reaction, a new stereogenic center was created, and the mixture of cis/trans 1,3‐disubstituted 1,2,3,4‐tetrahydro‐β‐carbolines was obtained. The ratio of cis/trans diastereomers depends on the stereogenic centre of used amino aldehyde and the size of substituents. It was confirmed by 1H and 2D NMR (ROESY) spectra. The conformations of cyclic products were studied by 2D NMR ROESY spectra. Products of the PS condensation after removal of protecting group(s) can be incorporated into a peptide chain as tryptophan mimetics with the possibility of the β‐turn induction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Atropisomerism in Rilpivirine hydrochloride: Spectroscopic characterization of Rilpivirine and related impurities

Nuclear magnetic resonance (NMR) technique has been used to find the property of atropisomerism in Rilpivirine hydrochloride by variable temperature analysis and various 2D techniques. Both the Rilpivirine hydrochloride (E‐isomer) and Impurity‐A (Z‐isomer) isomers have been differentiated and confirmed by NMR and ultraviolet techniques. Preparative high‐performance liquid chromatography isolation for Impurity‐A is followed by spectroscopic (NMR, mass spectra, and infrared) investigation that provides a perfect solution for determination of the structure of Rilpivirine and related impurities.     

Vibrational Circular Dichroism (VCD) Reveals Subtle Conformational Aspects and Intermolecular Interactions in the Carnitine Family

Vibrational circular dichroism spectra (VCD) in the mid‐IR region and electronic circular dichroism (ECD) spectra for three carnitine derivatives in the form of hydrochloride salts were recorded in deuterated methanol solutions. Density Functional Theory calculations help one to understand the significance of the observed VCD bands. VCD and ECD spectra are informative about the absolute configuration of the molecule, but VCD data reveal also some conformational aspects in the N,N,N‐trimethyl moiety and inform us about intermolecular interactions gained from the carbonyl stretching region for the acyl substituted carnitines. Chirality 27:907–913, 2015. © 2015 Wiley Periodicals, Inc.     

Axially chiral N‐(o‐aryl)‐4‐hydroxy‐2‐oxazolidinone derivatives from diastereoselective reduction of N‐(o‐aryl)‐2,4‐oxazolidinediones: Thermally interconvertible atropisomers via ring‐chain‐ring tautomerization

The reduction of the axially chiral N‐(o‐aryl)‐5,5‐dimethyl‐2,4‐oxazolidinediones by NaBH₄ yielded axially chiral N‐(o‐aryl)‐4‐hydroxy‐5,5‐dimethyl‐2‐oxazolidinone enantiomers having a chiral center at C‐4, with 100% diastereoselectivity as has been shown by their ¹H and ¹³C NMR spectra and by enantioselective HPLC analysis. The resolved enantiomeric isomers were found to interconvert thermally through an aldehyde intermediate formed upon ring cleavage via a latent ring‐chain‐ring tautomerization. It was found that the rate of enantiomerization depended on the size and the electronic effect of the ortho substituent present on the aryl ring bonded to the nitrogen of the heterocycle. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Stereolability of Chiral Ruthenium Catalysts With Frozen NHC Ligand Conformations Investigated by Dynamic‐HPLC

The stereolability of chiral Hoveyda–Grubbs II type ruthenium complexes bearing N‐heterocyclic carbene (NHC) ligands with Syn‐phenyl groups on the backbone and Syn‐ or Anti‐oriented o‐tolyl N‐substituents was studied by resorting to dynamic high‐performance liquid chromatography (D‐HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti‐ and two for Syn‐complexes) was achieved at very low temperatures (?53°C and ?40°C respectively), at which the NHC‐Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔG_isom^#), all derived by rotation around the NHC‐Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC‐Ru bond. Chirality 27:685–692, 2015. © 2015 Wiley Periodicals, Inc.     

N‐Acylated Alanine Methyl Esters (NAMEs) from Roseovarius tolerans,Structural Analogs of Quorum‐Sensing Autoinducers,N‐Acylhomoserine Lactones

The Roseobacter clade is one of the most important bacteria group living in the ocean. Liquid cultures of Roseovarius tolerans EL 164 were investigated for the production of autoinducers such as N‐acylhomoserine lactones (AHLs) and other secondary metabolites. The XAD extracts were analyzed by GC/MS. Two AHLs, Z7‐C14 : 1‐homoserine lactone (HSL) and C15 : 1‐HSL, were identified. Additionally, the extract contained five compounds with molecular‐ion peaks at m/z 104, 145, and 158, thus exhibiting mass spectra similar to those of AHLs with corresponding peaks at m/z 102, 143, and 156. Isolation of the main compound by column chromatography, NMR analysis, dimethyl disulfide derivatization for the determination of the location of the C?C bond and finally synthesis of the compound with the proposed structure confirmed the compound to be (Z)‐N‐(hexadec‐9‐enoyl)alanine methyl ester. Four additional minor compounds were identified as C14 : 0‐, C15 : 0‐, C16 : 0‐, and C17 : 1‐N‐acylated alanine methyl esters (NAMEs). All NAMEs have not been described from natural sources before. A BLASTp search showed the presence of AHL‐producing luxI genes, but no homologous genes potentially responsible for the structurally closely related NAMEs were found. The involvement of the NAMEs in chemical communication processes of the bacteria is discussed.     

Synthesis,Structural Characterization,and Chiroptical Studies of Bidentate Salen‐Type Lanthanide (III) Complexes

The salen‐type ligand prepared with (R,R) diphenylethan‐1,2‐diamine and salicylaldehyde provides stable and inert complexes KLnL₂ upon simple reaction with lanthanide halides or pseudohalides LnX₃ (Ln = Tb³⁺‐Lu³⁺; X = Cl^? or TfO^?) of its potassium salt. All the complexes were completely characterized through nuclear magnetic resonance (NMR), electronic circular dichroism (ECD) in the UV and some (Er³⁺, Tm³⁺, Yb³⁺) also with Near‐IR ECD (NIR‐ECD) and luminescence (Tb³⁺, Tm³⁺). Careful analysis of the NMR shifts demonstrated that the complexes are isostructural in solution and afforded an accurate geometry. This was further confirmed by means of Density Functional Theory (DFT) optimization of the Lu³⁺ complex, and by comparing the ligand‐centered experimental and time‐dependent TD‐DFT computed UV‐ECD spectra. As final validation, we used the NIR‐ECD spectrum of the Yb³⁺ derivative calculated by means of Richardson's equations. The excellent match between calculated and experimental ECD spectra confirm the quality of the NMR structure.  Chirality 27:857–863, 2015. © 2015 Wiley Periodicals, Inc.     

GMDP: unusual physico‐chemical and biological properties of the anomeriс forms

Disaccharide containing unit of peptidoglycan from bacterial cell wall, N‐acetyl‐d ‐glucosaminyl‐N‐acetylmuramyl‐l ‐alanyl‐d ‐glutaminamide (gluсosaminyl‐muramyl‐dipeptide) registered in Russia as an immunomodulatory drug, is shown to participate in slow equilibrium of α and β anomeric forms. Data of NMR spectra and molecular dynamics indicate that the α‐anomer predominantly acquires a folded conformation stabilized by intramolecular hydrogen bond between the alanyl carbonyl and muramyl NH proton. The β‐form displays a considerable fraction of extended, non‐hydrogen bonded structures. In the standard immunoadjuvant test system, the α‐form is practically inactive, and the activity of the equilibrium mixture with α : β = 68 : 32 ratio is due to the presence of β‐anomer. Such unique α–β selectivity of biological action must be considered at the design of related immunoactive glycopeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Zwitterionic Phosphorylated Quinines as Chiral Solvating Agents for NMR Spectroscopy

Because of their unique 3D arrangement, naturally occurring Cinchona alkaloids and their synthetic derivatives have found wide‐ranging applications in chiral recognition. Recently, we determined the enantioselective properties of C‐9‐phosphate mixed triesters of quinine as versatile chiral solvating agents in nuclear magnetic resonance (NMR) spectroscopy. In the current study, we introduce new zwitterionic members of this class of molecules containing a negatively charged phosphate moiety (i.e., ethyl, n‐butyl and phenyl hydrogen quininyl phosphate). An efficient approach for synthesizing these compounds is elaborated, and full characterization, including conformational and autoaggregation phenomena studies, was performed. Therefore, their ability to induce NMR anisochrony of selected enantiomeric substrates (i.e., primarily N‐DNB‐protected amino acids and their methyl esters) was analyzed compared to uncharged diphenyl quininyl phosphate and its positively charged quaternary ammonium hydrochloride salt. In addition, ¹H and ¹³C NMR experiments revealed their enantiodiscrimination potential toward novel analytes, such as secondary amines and nonprotected amino acids. Chirality 27:752–760, 2015. © 2015 Wiley Periodicals, Inc.     

A Simple 13C NMR Method for the Discrimination of Complex Mixtures of Stereoisomers: All Eight Stereoisomers of α‐Tocopherol Resolved

A simple one‐dimensional ¹³C NMR method is presented to discriminate between stereoisomers of organic compounds with more than one chiral center. By means of this method it is possible to discriminate between all eight stereoisomers of α‐tocopherol. To achieve this the chiral solvating agent (S)‐(+)‐1‐(9‐anthryl)‐2,2,2‐trifluoroethanol and the compound of interest were dissolved in high concentrations in chloroform‐d, and the nuclear magnetic resonance (NMR) spectrum was recorded at a low temperature. The individual stereoisomers of α‐tocopherol were assigned by spikes of the reference compounds. The method was also applied to six other representative examples. Chirality 27:850–855, 2015. © 2015 Wiley Periodicals, Inc.     

Flow‐injection chemiluminescence determination of diazepam by oxidation with N‐bromosuccinimide

A rapid and sensitive flow‐injection chemiluminescence (FI–CL) method is described for the determination of diazepam based on its reaction with N‐bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy‐transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10^?6 to 2.0 × 10^?4 mol/L with a detection limit of 5.0 × 10^?7 mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10^?5 mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Stereochemistry of the Brivaracetam Diastereoisomers

The stereochemistry of all four stereoisomers of brivaracetam was determined using vibrational circular dichroism (VCD) spectroscopy. By comparing experimentally obtained VCD spectra and computationally simulated ones, the absolute configurations can be confidently assigned without prior knowledge of their relative stereochemistry. Neither the corrected mean absolute errors analysis of the nuclear magnetic resonance (NMR) data, nor the matching of experimental and calculated infrared spectra allowed the diastereoisomers to be distinguished. VCD spectroscopy itself suffices to establish the absolute configurations of all diastereoisomers. The relative stereochemistry could also be statistically confirmed by matching experimental and computed NMR spectra using the CP3 algorithm. The combination of VCD and NMR is recommended for molecules bearing more than one chiral center, as the relative configurations obtained from NMR serve as an independent check for those established with VCD. Analysis of the calculated VCD spectra reveals that the localized NH₂ scissoring mode at around 1600 cm^‐1 is characteristic for intramolecular hydrogen bonding, while the orientation of the ethyl group is reflected by the delocalized modes between 1150 and 1050 cm^‐1. Chirality 28:215–225, 2016. © 2016 Wiley Periodicals, Inc.     

Bifunctional Platinum(II) Complexes with Bisphosphonates Substituted Diamine Derivatives: Synthesis and In vitro Cytotoxicity

A series of N,N′‐dibisphosphonate‐containing 1,3‐propanediamine derivatives ( L1 – L6 ) and their corresponding dichloridoplatinum(II) complexes ( 1 – 6 ) have been synthesized and characterized by elemental analysis, ¹H‐NMR, ¹³C‐NMR, ³¹P‐NMR and HR‐MS spectra. The in vitro antitumor activities of compounds L1 – L6 and 1 – 6 were tested by WST‐8 assay with Cell Counting Kit‐8, indicating that platinum‐based complexes 1 – 6 showed higher cytotoxicity than corresponding ligands L1 – L6 against A549 and MG‐63, especially complex 2 which displayed comparable cytotoxicity to those of cisplatin and zoledronate after 48 h incubation. In addition, complexes 1 – 6 were more active in vitro on osteosarcoma cell line MG‐63 than normal osteoblast cell line hFOB 1.19. The structure‐activity relationship has been summarized based on the in vitro cytotoxicity of three series of platinum complexes from this and our previous studies. The in vitro bone affinity of platinum complexes was also tested by hydroxyapatite (HAP) chromatography in terms of capacity factor K′. Besides, in this paper, representative complex 2 , which has been proved to be a promising antitumor agent with high cytotoxicity and bone HAP binding property, was investigated for its mechanism of action producing cell death against MG‐63.     

Nuclear magnetic resonance spectra of poly(N-alkylamino acid)s

220-MHz NMR spectra of various poly (N-alkylamino acid)s are investigated. Spectra of polysarcosine recorded in various solvents showed fine splittings of the methyl and methylene bands. Comparing the spectrum with that of its model compound, the fine structure of the methyl band of polysarcosine was assigned to four dyad sequences of the cis–trans isomeric state of the main chain amide bonds. Also the methylene band was roughly divided into cis and trans bands. From the temperature dependence of the spectra of polysarcosine, a double coalescence phenomenon was observed, in which the four dyad peaks coalesced into two peaks corresponding to cis and trans, then the two peaks coalesced into one peak. Further, the approximate value of the free energy for the internal rotation of the main chain amide bond was estimated. NMR spectra of various poly(N-alkylglycine)s in methylene chloride solution were also obtained. From the comparsion of their methylene bands, the introduction of the bulky N-alkyl groups was found to increase the cis content of the amide bond.     

A highly selective colorimetric and fluorescent dual‐modal probe for the rapid determination of fluoride anions

In this work, 4‐(p ‐hydroxybenzylidenehydrazino)‐N ‐butyl‐1,8‐naphthalimide ( 1 ) has been designed and synthesized as a colorimetric and fluorescent dual‐modal probe for F^?. Compound 1 immediately detected inorganic fluoride salts using UV /v is absorption and fluorescence spectroscopy methods, and served as a ‘naked‐eye’ indicator for F^? with high selectivity and sensitivity. Both the absorption and fluorescence spectra show excellent linearity with the concentration of F^?. Real‐life applications demonstrated that our proposed analytical system provided a satisfactory method for the determination of F^?. In addition, the reaction mechanism of deprotonation was confirmed by ¹H NMR.     

Site‐specific labeling of synthetic peptide using the chemoselective reaction between N‐methoxyamino acid and isothiocyanate

Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Total synthesis and stereochemical reassignment of tasiamide B

The first total synthesis of tasiamide B, an octapeptide bearing 4‐amino‐3‐hydroxy‐5‐phenylpentanoic acid unit isolated from the marine cyanobacteria Symploca sp. is described. A simple and efficient way was found to avoid the pyroglutamylation of N^α‐Me‐Gln and led to a reassignment of the N^α‐Me‐L ‐Phe of tasiamide B to be N^α‐Me‐D ‐Phe, which was also supported by 1D and 2D NMR. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012