Sulfate esters of morphine derivatives: synthesis and characterization.

Sixteen 3-O- and 6-O-sulfate esters of morphine, codeine and some of their N-methyl quaternary derivatives were synthesized by means of sulfation with pyridine-SO(3) complex and sulfuric acid/N,N'-dicyclohexylcarbodiimide. Complete (1)H- and (13)C-NMR assignments are given for each of the synthesized compounds based on one- and two-dimensional homo- and heteronuclear measurements. Comparative analysis of chiral properties by circular dichroism and optical rotatory dispersion revealed characteristic differences in the spectra due to changes in charge, polarity and intramolecular association by strong hydrogen bonds in aqueous solution. The synthesized sulfate esters are prospective peripheral analgesics lacking central side effects and are also useful as reference substances for various analytical studies involving sulfate ester metabolites.

Structure and stereochemistry of novel ecdysteroids from the roots of Serratula wolffii.

Three new natural ecdysteroids viz. 22-dehydro-20-deoxy-ajugasterone C (1), 1-hydroxy-22-deoxy-20,21-didehydro-ecdysone (2) and 22-deoxy-20,21-didehydro-ecdysone (3) were isolated from the methanol extract of the roots of Serratula wolffii. The structures of compounds 1-3 were established by various spectroscopic techniques, including one- and two-dimensional NMR, circular dichroism and mass spectroscopic methods.

Gradient HPLC separation of dehydroepiandrosterone (DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in the chromatographic mechanism.

A three-step gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the separation of dehydroepiandrosterone (DHEA), its sulfate ester (DHEA-S), its three C7-oxidized metabolites (7alphaOH-DHEA, 7betaOH-DHEA, 7-keto-DHEA), and its biosynthetic congeners (androstenedione, testosterone, estradiol, pregnenolone). This new method allows the quantitative characterization of DHEA metabolism and biosynthetic transformation under given physiological, pathological, or therapeutically influenced circumstances. Tetrahydrofuran probably acts as a proton acceptor coadsorbent, while isopropanol behaves as a proton donor during the separation of testosterone, estradiol, and the stereoisomers of 7-OH-DHEA.

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode.

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewinski competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.

Utility of cyclodextrins in the formulation of genistein part 1. Preparation and physicochemical properties of genistein complexes with native cyclodextrins.

Isoflavones are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs). The molecular encapsulation with CDs results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of isoflavones. Genistein, a key isoflavone constituent of Ononidis spinosae radix was found to form a supramolecular, non-covalent inclusion complex with both beta-cyclodextrin (beta-CD) and gamma-cyclodextrin (gamma-CD), while it did not form a stable complex with alpha-CD. The guest genistein was found to spatially located in the less polar cavity of cyclodextrin. The isolated binary genistein/CD complexes appeared novel crystalline lattices. The in vitro dissolution of genistein entrapped into both beta- and gamma-CD, significantly surpassed that of the plain isoflavone.

Three new steroids from the roots of Serratula wolffii.

Investigation of the methanol extract of the roots of Serratula wolffii resulted in an ecdysone-related compound, 2beta,3beta,20R,22R,25-pentahydroxy-5beta-cholest-6,8(14)-dien (1), a new ecdysteroid, 24-methylene-shidasterone (2), the known compound stachysterone B (3) and its 14,15-alpha-epoxide (4), a novel natural product. The structures of compounds 1-4 were established by spectral analysis ((1)H NMR, (13)C NMR, COSY, NOESY, HMQC, HMQC-TOCSY and HMBC).

Preparative-scale chromatography of ecdysteroids of Serratula wolffli andrae.

Numerous ecdysteroids are isolated from the herb of Serratula wolffii Andrae, a cultivated plant. The isolation procedure includes a variety of low-pressure liquid chromatography, thin-layer chromatography (TLC), gel chromatography, and high-performance liquid chromatography (HPLC) methods. The progress of separation is monitored by TLC, and the final proof of purity is carried out by HPLC. The isolation process involves the removal of proteins, flavonoids, chlorophylls, other sterines, etc. The purification also includes the separation of the target ecdysteroids from each other. Isolation of the pure compounds requires 2-8 chromatographic steps. The consecutive steps are based on the different physicochemical properties of the ecdysteroids. In some cases, a special peak-cut method employing a flush of dichloromethane into the dichloromethane-isopropanol-water mobile phase is used. This flush of dichloromethane leads to an almost perfect separation of otherwise unresolved peaks. Two ecdysteroids, 25-hydroxydacryhainansterone and 14-epi-20-hydroxyecdysone, are identified as natural products for the first time. The structure-chiroptical relationships for some ecdysteroids are also discussed.

Tracking of enantioselective solubility of rac-norgestrel in the presence of cyclodextrin by a CD spectroscopic method.

Dissolution of hydrophobic rac-norgestrel in aqueous gamma-cyclodextrin (gamma-CyD) and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CyD) solutions was investigated, and enantioselective dissolution was observed. (-)-Norgestrel, the eutomer molecule, was dissolved to a greater extent using each of the CyDs, although the effect was more significant in the case of HP-gamma-CyD. A circular dichroism (CD) spectroscopic method based on measurement of the anisotropy factor was applied for the determination of the enantiomer ratio. The concentration and the enantiomer ratio of norgestrel were determined indirectly in octanol after extraction. Optical rotation dispersion (ORD) measurements could confirm that neither the free CyDs nor their inclusion complexes could get into the organic phase during transport to octanol. Only the norgestrel molecules were able to get into the organic phase, although the enantiomer ratio remained the same as was obtained in the aqueous CyD solution.

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases.

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.

[Ecdysteroids of Silene italica ssp. nemoralis, novel approaches of ecdysteroid therapy]. / Ekdiszteroidok a Silene italica ssp. nemoralis-ból, az ekdiszteroidok felhasználásának néhány uj lehetosége.

Ecdysteroids are known as insect moulting hormones. They have the basic steroid structure, although their physiological effects on mammalians do not show the thymolytic and androgenic side effects of vertebrate type steroid hormones. At the same time, phytoecdysteroids can be used utilizing their anabolic and adaptogenic effects. Ecdysteroids also have a tremendous potential in the most modern therapy. Recently, the biotechnology started to employ ecdysteroids as powerful inducers for gene-switch systems with insertion of modified insect receptor into the malignant cells. Nineteen ecdysteroids were isolated with combined chromatographic methods from the herbs of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The chemical structure of the isolated compounds have been elucidated using spectroscopic methods (x-ray, UV, CD, IR, MS, 1D-, 2D-NMR, HMQC, HMBC, COSY, TOCSY, NOESY, ROESY). Structural determination of two of the five new ecdysteroids is detailed here.

[Enantioseparation of quinazolone derivatives using an AGP-based liquid chromatography stationary phase]. / Az AGP-alapú folyadék-kromatográfiás állófázis alkalmazása kinazolon származékok enantiomerjeinek elválasztásában.

An AGP-based chiral stationary phase has been applied successfully for enantioseparation of 4(3H)-quinazolone derivatives. Chiral-AGP is proved to be an excellent selector, as optimized chromatographic conditions allow with one exception baseline resolution for the enantiomers of the potential cholecystokinin antagonist compounds (alpha = 1.19-1.85). Retention and enantioselectivity could be modified to a large extent by varying the eluent pH and adding organic solvents with different types i.e. acetonitrile and 2-propanol to the buffered mobile phase. It was established that by increasing the eluent pH from 6.0 to 7.0 the retention factors of of the model compounds bearing no protonable groups are increased in the presence of 7 v/v % (1.33 M) acetonitrile. However further increasing the acetonitrile content up to 10 v/v % or addition of 2-propanol in equimolar concentration (1.33 M) no similar changes could be detected with the same modification in the eluent pH. These observations are explained by changes in the sorption properties of the selector determined simultaneously by the type and concentration of organic modifier and also the eluent pH. The experimental data give further insight into the chromatographic mechanism on a Chiral-AGP column.

Study on the sorption properties of alpha1-acid glycoprotein (AGP)-based stationary phase modified by organic solvents.

An alpha(1)-acid glycoprotein, immobilized on silica (Chiral-AGP) is one of the most widely used chiral stationary phases for the enantiomeric separation of a wide variety of chiral drugs with several applications in the biological and clinical field. The aim of this work was to study the sorption properties of the AGP-based stationary phase, which may have crucial importance for enantioselectivity. New binding data to the mechanism of the chromatographic separation are presented. The sorption of both organic solvents, i.e., acetonitrile and dioxane, shows remarkable pH dependency. A fluorescence quenching study was carried out to elucidate structural changes of AGP in the presence of acetonitrile using 2,2,2-trichloroethanol as fluorescence quencher.

Synthesis and stereochemistry of dispiro substituted pyridazines: application of ellipticity-absorbance ratio spectra for proving enantiomeric relationship by HPLC-CD/UV detection.

The aim of our study was to synthesize vinylic and pyrido-fused pyridazines with a spirano moiety and to investigate their stereochemistry by spectroscopic and HPLC analyses. The vinylic compounds 5 were obtained by Knoevenagel condensation of cyclohexylidene malonates 1 with pyridazinecarbaldehyde 2. Compound 5b exhibits geometric isomerism identified by NMR, HPLC, and X-ray methods. The thermal rearrangement reactions of compounds 5 easily led to the pyridopyridazine derivatives 6. In the case of 6b, possessing both central and axial chirality, both diastereomers and the respective enantiomers were detected. Their stereochemical relationships could be determined by HPLC-CD/UV analyses with application of anisotropy spectra in a novel way.