Liquid chromatographic resolution of 3-amino-1,4-benzodiazepin-2-ones on crown ether-based chiral stationary phases

3-Amino-5-phenyl (or 5-methyl)-1,4-benzodiazepin-2-ones, which are chiral precursors of anti-respiratory syncytial virus active agents, were resolved on three different chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid or (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6. Among the three CSPs, the CSP that is based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 and containing residual silanol group-protecting n-octyl groups on the silica surface was found to be most effective with the use of 80% ethanol in water containing perchloric acid (10 mM) and ammonium acetate (1.0 mM) as a mobile phase. The separation factors (α) and resolutions (R(S) ) were in the range of 1.90-3.21 and 2.79-5.96, respectively. From the relationship between the analyte structure and the chromatographic resolution behavior, the chiral recognition mechanism on the CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was proposed to be different from that on the CSP based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6. In addition, the chromatographic resolution behavior of the most effective CSP was investigated as a function of the composition of aqueous mobile phase containing organic and acidic modifier and ammonium acetate.     

Amino acid-based enantiomerically pure 3-substituted 1,4-benzodiazepin-2-ones: a new class of anti-ischemic agents

A series of 3-substituted 1,4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ischemic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling studies.     

Synthesis and HPLC chiral recognition of regioselectively carbamoylated cellulose derivatives

Four regioselective-carbamoylated cellulose derivatives having two different substituents at 2-, 3-, and 6-position were prepared and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. Investigations showed that the nature and arrangement of the substituents significantly influenced the chiral recognition abilities of the heterosubstituted cellulose derivatives and each derivative exhibited characteristic enantioseparation. Some racemates were better resolved on these derivatives than the corresponding homogeneously substituted cellulose derivatives including a commercial CSP, Chiralcel OD. Racemic compounds shown in this study were most effectively discriminated on cellulose 2,3-(3-chloro-4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) and 2,3-(3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate).     

A convenient and validated enantiomer separation of chiral aliphatic amines as nitrobenzoxadiazole derivatives on polysaccharide‐derived chiral stationary phases under simultaneous ultraviolet and fluorescence detection

A convenient method using a fluorogenic agent, 4‐chloro‐7‐nitro‐1,2,3‐benzoxadiazole (NBD‐Cl), was developed for enantiomer separation of chiral aliphatic amines including amino alcohols by normal high‐performance liquid chromatography. The enantiomer separation of chiral aliphatic amines as NBD derivatives was performed on six covalently bonded and four coated‐type polysaccharide‐derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence detection (FLD). Among the covalently bonded CSPs, Chiralpak IE showed the best enantiomer separation for most analytes. The other CSPs also showed good enantioselectivity except for Chiralpak IB. On the other hand, Chiralpak AD‐H and Amylose‐1 generally exhibited better enantiomer separation of NBD derivatized chiral amines among the coated CSPs. The developed analytical technique was also applied to determine the optical purity of commercially available (R)‐ and (S)‐leucinol; the impurity was found to be 0.06%. The developed method was validated and proved to be an accurate, precise, sensitive, and selective method suitable for separation of chiral aliphatic amines as NBD derivatives under simultaneous UV and FLD.     

Enantioseparation using urea- and imide-bearing chitosan phenylcarbamate derivatives as chiral stationary phases for high-performance liquid chromatography

Completely deacetylated chitosan was prepared by the treatment of commercial chitosan with 50% aqueous NaOH, and then derivatized into several new chitosan phenylcarbamate derivatives having a urea and an imide moiety at the 2-position of the glucosamine ring by the reaction with isocyanate and phthalic anhydride/isocyanate, respectively. The chitosan derivatives were coated on macroporous silica gel and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The chiral recognition ability of the chitosan derivative was improved using the completely deacetylated chitosan. Among the novel chitosan derivatives, the 3,5-dimethyl-, 3,5-dichloro-, and 3,4-dichlorophenylcarbamate derivatives were found to possess relatively high chiral resolution abilities. The CSPs based on the chitosan phenylcarbamate-urea and -imide derivatives were stable in the presence of chloroform and ethyl acetate as a component of the eluents, and some racemates were better resolved by such eluents. The dichlorophenylcarbamate-imide derivatives showed a high chiral recognition for metal acetylacetonate complexes. The enantiomerization of Al(acac)3 was performed on the chitosan 3,5-dichlorophenylcarbamate-imide derivative CSP and the resulting chromatogram showed a 26% (+)-isomer enrichment.     

Enantiomers of C(5)-chiral 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives: Analytical and semipreparative HPLC separation, chiroptical properties, absolute configuration, and inhibitory activity against monoamine oxidase

The HPLC enantiomer separation of a novel series of C(5)-chiral 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives, with inhibitory activity against monoamine oxidases (MAO) type A and B, was accomplished using polysaccharide-based chiral stationary phases (CSPs: Chiralpak AD, Chiralcel OD, and Chiralcel OJ). Pure alcohols, such as ethanol and 2-propanol, and typical normal-phase binary mixtures, such as n-hexane and alcohol modifier, were used as mobile phases. Single enantiomers of several analytes examined were isolated on a semipreparative scale, and their chiroptical properties were measured. The assignment of the absolute configuration was established for one compound by single-crystal X-ray diffraction method and for the other three by CD spectroscopy. The inhibitory activity against MAO of racemic samples and single enantiomers were evaluated in vitro.     

Stereochemical features of 1,4-benzodiazepin-2-ones bound to human serum albumin: difference CD and UV studies

The stereochemistry of an achiral (Diazepam) and two chiral (3-methyl and 3-succinyloxy substituted) 1,4-benzodiazepin-2-ones interacting with human serum albumin (HSA) has been investigated by making use of difference absorption (UV) and circular dichroism (CD) spectroscopies. Evidence is obtained for a higher affinity with HSA for one of the two possible conformations of the seven-membered benzodiazepine ring. The red shift revealed by the absorption difference spectrum between the free and the bound drug accounts for the CD difference spectra observed.     

Liquid chromatographic resolution of N-acyl-alpha-amino acids as their anilide derivatives on a chiral stationary phase based on (S)-leucine

A chiral stationary phase (CSP 1) derived from N-(3,5-dinitrobenzoyl)leucine N-phenyl N-alkylamide was used for the liquid chromatographic resolution of anilide derivatives of N-acyl-alpha-amino acids and the chromatographic resolution results were compared with those from four other commercial CSPs. The chromatographic resolution results showed that CSP 1 was most effective among five CSPs used in this study. The chiral recognition mechanism exerted by CSP 1 for the resolution of anilide derivatives of N-acyl-alpha-amino acids is proposed to involve a face-to-face pi-pi interaction and two hydrogen bonding interactions between the CSP and the analytes from the chromatographic resolution behaviors of slightly modified anilide derivatives of N-acyl-alpha-amino acids. The chiral recognition mechanism proposed is quite similar to that advanced previously for the resolution of N-(3,5-methoxybenzoyl)-alpha-amino acids on CSP 1, even though the interaction sites of the two types of analytes were totally different from each other. The apparent similarity of the two chiral recognition mechanisms was assumed to stem from the identical interaction modes of the two types of analytes with the CSP. In addition, the dependence of the enantioselectivity of anilide derivatives of N-acyl-alpha-amino acids on the length of the alkyl tail of the N-acyl group of analytes was rationalized to stem from the intercalation of the N-acyl group of the (R)-enantiomer of analytes between the tethers of the CSP.     

The comparison in enantioseparation ability of the chiral stationary phases with single and mixed selector--the selectors derived from two D-tartrates

(2S,3S)-2,3-Bis(3,5-dimethylphenylcarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid and (2S,3S)-2,3-bis(1-naphthalenecarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid were synthesized from D-tartaric acid. These two compounds were chlorinated to afford two chiral selectors for chiral stationary phases (CSPs). The selectors were separately immobilized on aminated silica gel to give two single selector CSPs; and were simultaneously immobilized to obtain a mixed selector CSP. Comparing to the single selector CSPs, the mixed selector CSP bears the enhanced enantioseparation ability, suggesting that the two selectors in the mixed selector CSP are consistent for chiral recognition in most mobile phase conditions.     

Analysis and purification of alcohol-sensitive chiral compounds using 2,2,2-trifluoroethanol as a modifier in supercritical fluid chromatography

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities (alpha) and resolutions (Rs) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.     

Immobilization of difunctional building blocks on hydroxysuccinimide activated silica: versatile in situ preparation of chiral stationary phases.

Several brush-type chiral stationary phases (CSPs) based on undecanoyl- or butanoyl-bound (R,R)-1,2-diphenylethane-1,2-diamine (DPEDA) as chiral selector were prepared by an innovative, fast, and less expensive kind of preparation. The key to this method is the immobilization of the enantiomeric pure diamine with only one amino function in a simple substitution reaction on hydroxysuccinimide ester-activated silica. No excess chiral material is lost. Loading can be easily monitored analyzing the filtrate. The free second amino function can subsequently be acylated with different acyl halogenides. Examples with benzoyl- and 3,5-dinitrobenzoyl (DNB) amides show that, based on our new approach, a library of differently acylated Pirkle-type CSPs can easily be obtained. A benzoylated analog of the commercially available ULMO CSP is shown to be very effective in separating enantiomers of N-acyl amino acids.     

Direct separation of albendazole sulfoxide enantiomers by liquid chromatography on a chiral column deriving from (S)-N-(3,5-dinitrobenzoyl) tyrosine: application to enantiomeric assays on plasma samples

The direct enantiomeric resolution of albendazole sulfoxide (SOABZ), an anthelmintic drug belonging to the benzimidazole class, is reported on a chiral stationary phase (CSP) synthesized by covalent binding of (S)-N-(3,5-dinitrobenzoyl)tyrosine-O-(2-propen-1-yl) methyl ester on a gamma-mercaptopropyl-silanized silica gel. A comparison with the resolution achieved on commercially available Pirkle-type CSPs obtained from N-(3,5-dinitrobenzoyl) derivatives of (R)-phenyglycine or (S)-phenylalanine is described. Some structurally related chiral sulfoxides including oxfendazole (SOFBZ) are also studied. Optimization of the mobile phase nature and composition is investigated showing that a hexane-dioxane-ethanol ternary mixture affords an almost baseline resolution (Rs = 1.25); however, in this case, albendazole sulfone (SO2ABZ) is eluted between the two sulfoxide enantiomers; accordingly, a hexane-ethanol mobile phase would be preferred for biological samples containing both metabolites. The influence of temperature on the resolution is depicted with a hexane-ethanol mobile phase. Finally, application to the enantiomeric assays of SOABZ in plasmatic extracts of rat, sheep, bovin, and man after oral administration of albendazole (sulfoxidized to SOABZ and SO2ABZ) is reported. Some distortions in the enantiomeric ratios are evidenced depending on the species.     

Novel chiral stationary phases based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin combining cinchona alkaloid moiety

Novel chiral selectors based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin connecting quinine (QN) or quinidine (QD) moiety were synthesized and immobilized on silica gel. Their chromatographic performances were investigated by comparing to the 3,5-dimethyl phenylcarbamoylated β-cyclodextrin (β-CD) chiral stationary phase (CSP) and 9-O-(tert-butylcarbamoyl)-QN-based CSP (QN-AX). Fmoc-protected amino acids, chiral drug cloprostenol (which has been successfully employed in veterinary medicine), and neutral chiral analytes were evaluated on CSPs, and the results showed that the novel CSPs characterized as both enantioseparation capabilities of CD-based CSP and QN/QD-based CSPs have broader application range than β-CD-based CSP or QN/QD-based CSPs. It was found that QN/QD moieties play a dominant role in the overall enantioseparation process of Fmoc-amino acids accompanied by the synergistic effect of β-CD moiety, which lead to the different enantioseparation of β-CD-QN-based CSP and β-CD-QD-based CSP. Furthermore, new CSPs retain extraordinary enantioseparation of cyclodextrin-based CSP for some neutral analytes on normal phase and even exhibit better enantioseparation than the corresponding β-CD-based CSP for certain samples.     

Structure-activity study of 2,3-benzodiazepin-4-ones noncompetitive AMPAR antagonists: identification of the 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one as neuroprotective agent

In the search for AMPA receptor (AMPAR) antagonists, 2,3-benzodiazepines represent a family of specific noncompetitive antagonists with anticonvulsant and neuroprotective properties. We have previously shown that 2,3-benzodiazepin-4-ones possess marked anticonvulsant properties and high affinity for the noncompetitive binding site of the AMPAR complex. In this paper, we report the synthesis and pharmacological characterization of a full set of 2,3-benzodiazepin-4-ones in order to better define the structure-activity relationship (SAR) of this class of compounds. Binding assays and functional tests were performed to evaluate the antagonistic activity at the AMPARs. Through these results we have identified a potent AMPAR antagonist, 1-(4-amino-3-methylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-one (5c). This compound noncompetitively inhibited AMPAR-mediated toxicity in primary mouse hippocampal cultures with an IC(50) of 1.6muM and blocked kainate-induced calcium influx in rat cerebellar granule cells with an IC(50) of 6.4muM. Thus, 5c has the in vitro potential as therapeutic drug in the treatment of various neurological disorders.     

The Seven Carbohydrate Bioengineering Meeting,Braunschweig, Germany,April, 22–25, 2007

Stereocontrol in bakers' yeast reduction can be achieved by introduction of a sulfur functional group into substrates. α-Methylthio-β-keto esters are reduced to give exclusively (3S)-3-hydroxy esters. α-Substituted β-keto thiol esters and dithioesters afford (2R,3S)-3-hydroxy esters with high diastereo-and enantioselectivity. Ketones possessing 1,3-dithiane, phenylsulfenyl, or phenylsulfonyl groups at the α-position are transformed also into the corresponding (S)-secondary alcohols. Optically pure (S)-(phenylsulfinyl)acetones can be obtained by kinetic resolution of racemic derivatives with the yeast. Diastereo- and enantioselective reduction of 1,2-diketones leading into (1S,2S)-1,2-diol derivatives can be also achieved by introduction of 1,3-dithiane, phenylsulfenyl or phenylsulfonyl groups into the α-position. Reductions of carbon-carbon double bond of sulfur-functionalized prenyl derivatives provide both chiral (R)- and (S)-C₅-building blocks for terpenoid synthesis. The utility of the reduction products as chiral building blocks is demonstrated in the synthesis of biologically active natural products such as pheromones, sugars, antibiotics etc. by functional group transformation and carbon-carbon bond formation reactions with the aid of sulfur functional groups.     

Synthesis and anti-HIV activity of 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-one (TBO) derivatives. Truncated 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2( 1H)-on es (TIBO) analogues

4,5,6,7-Tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-ones (TIBO), 1, have been shown to significantly inhibit HIV-1 replication, as reported in detail in our prior publications. Since our earlier reports, we have modified the TIBO structures 1 by removing the 5-membered ring of 1, generating 1,3,4,5-tetrahydro-2H-1,4-benzodiazepin-2-ones (TBO), 4, a bicyclic series of compounds. Although compounds 4 possess modest activity when compared to TIBO analogues 1, they clearly demonstrated significant anti-HIV-1 activity.     

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones as potent and selective I(Ks)-blocking class III antiarrhythmic agents

Novel 5-cyclopropyl-1,4-benzodiazepin-2-ones having various N-l substituents were identified as potent and selective blockers of the slowly activating cardiac delayed rectifier potassium current (I(Ks)). Compound 11 is the most potent I(Ks) channel blocker reported to date.     

Enantiomerization of 3-carbethoxy-l,4-benzodiazepin-2-one: combined chiral HPLC and spectroscopic study.

Recently developed chiral HPLC columns CHIRIS AD1 and CHIRIS AD2 have been demonstrated to separate racemic, configurationally unstable ethyl-7-chloro-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepine-3-carboxylate (1) and its 3-methyl congener 2; fast on-column enantiomerization of configurationally unstable 1 was observed, however. Addition of 0.1% of AcOH to the eluting mixture inhibits enantiomerization, whereas the same percentage of Et(3)N completely precludes enantioseparation, suggesting base-catalysis by free beta-aminoethyl groups, present in low percentage in chiral stationary phase (CSP). When both CSPs were prepared under conditions of nonexhaustive acylation by N-DNB-alpha-aminoacids, no separation of 1 was observed. The rate of enantiomerization on CHIRIS AD2 was determined at 25 degrees C, the mechanism is discussed, and experimental results correlated with calculated relative stabilities of the tautomers la-c. Absolute (3S) configuration of (+) enantiomers of 1 and 2 was determined by comparison of their eluation profile to that of (+/-)-3 and (3S)-(+)-3, taking into account relative (psia or psie) configuration of the prevailing conformer in solution.     

Chiral separation on various modified amino alcohol‐derived HPLC chiral stationary phases

3,5‐Dinitrobenzoyl chloride was previously used for the preparation of (R)‐phenylglycinol‐ and (S)‐leucinol‐derived chiral stationary phases. In this study, 3,5‐bis(trifluoromethyl)benzoyl chloride, 2‐furoyl chloride, 2‐theonyl chloride, 10,11‐dihydro‐5H‐dibenzo[b,f]azepine‐5‐carbonyl chloride, diphenylcarbamoyl chloride, and 1‐adamantanecarbonyl chloride were used to prepare six new phenylglycinol‐derived chiral stationary phases (CSPs) and five new leucinol‐derived CSPs. Using these 11 CSPs, chiral separation of nine π‐acidic amino acid derivatives and five π‐basic compounds was performed, and the separation results were compared. An adamantyl‐derived CSP showed good separation. Chirality 28:276–281, 2016. © 2016 Wiley Periodicals, Inc.     

Effect of substituents of the benzoquinone ring on electron-transfer activities of ubiquinone derivatives

The effect of substituents on the 1,4-benzoquinone ring of ubiquinone on its electron-transfer activity in the bovine heart mitochondrial succinate-cytochrome c reductase region is studied by using synthetic ubiquinone derivatives that have a decyl (or geranyl) side-chain at the 6-position and various arrangements of methyl, methoxy and hydrogen in the 2, 3 and 5 positions of the benzoquinone ring. The reduction of quinone derivatives by succinate is measured with succinate-ubiquinone reductase and with succinate-cytochrome c reductase. Oxidation of quinol derivatives is measured with ubiquinol-cytochrome c reductase. The electron-transfer efficacy of quinone derivatives is compared to that of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone. When quinone derivatives are used as the electron acceptor for succinate-ubiquinone reductase, the methyl group at the 5-position is less important than are the methoxy groups at the 2- and 3-positions. Replacing the 5-methyl group with hydrogen causes a slight increase in activity. However, replacing one or both of 2- and 3-methoxy groups with a methyl completely abolishes electron-acceptor activity. Replacing the 3-methoxy group with hydrogen results in a complete loss of electron-acceptor activity, while replacing the 2-methoxy with hydrogen results in an activity decrease by 70%, suggesting that the methoxy group at the 3-position is more specific than that at the 2-position. The structural requirements for quinol derivatives to be oxidized by ubiquinol-cytochrome c reductase are less strict. All 1,4-benzoquinol derivatives examined show partial activity when used as electron donors for ubiquinol-cytochrome c reductase. Derivatives that possess one unsubstituted position at 2, 3 or 5, with a decyl group at the 6-position, show substrate inhibition at high concentrations. Such substrate inhibition is not observed when fully substituted derivatives are used. The structural requirements for quinone derivatives to be reduced by succinate-cytochrome c reductase are less specific than those for succinate-ubiquinone reductase. Replacing one or both of the 2- and 3-methoxy groups with a methyl and keeping the 5-position unsubstituted (plastoquinone derivatives) yields derivatives with no acceptor activity for succinate-Q reductase. However, these derivatives are reducible by succinate in the presence of succinate-cytochrome c reductase. This reduction is antimycin-sensitive and requires endogenous ubiquinone, suggesting that these (plastoquinone) derivatives can only accept electrons from the ubisemiquinone radical at the Qi site of ubiquinol-cytochrome c reductase, and cannot accept electrons from the QPs of succinate-ubiquinone reductase.