The role of harmonized, gas and liquid chromatography mass spectrometry in the discovery of the neolignan balanophonin in the fruit wall of Cirsium vulgare.

In order to identify and quantify fruit-lignans of Cirsium vulgare - authors introduced a special analysis system: with particular attention to the lignans enrichment/separation course. These synchronized, germination and enzymatic hydrolysis processes were followed by complementary gas and liquid chromatography, coupled with special mass selective detections (GC-MS, LC-MS/MS, LC-TOF/MS) and confirmed by nuclear magnetic resonance (NMR) spectroscopy. Mass fragmentations and NMR evidences, proved that the two main medicinal lignan constituents of the fruits of Cirsium vulgare are the neolignan-type, free balanophonin and the butyrolactone-type tracheloside. As novelty to the field, these two lignans of different chemical structures could be quantitatively extracted, separately from each others, without impurities. Balanophonin and tracheloside do accumulate in the fruits of C. vulgare, separately: balanophonin was found, in enormous high concentrations, in the fruit wall (23.2-24.9 mg/g), while in embryo part tracheloside was determined (20.3mg/g), exclusively. Consequently, the optimum source of balanophonin proved to be the fruit wall, while tracheloside, - providing trachelogenin upon enzymatic hydrolysis, - could be obtained from the embryo parts of fruits. As further novelties of the study balanophonin was identified and quantified at the first time with on-line chromatographic technique, in free form, without authentic standard compound.

A novel µ-opioid receptor ligand with high in vitro and in vivo agonist efficacy.

The aims of this study were to synthesize 14-O-Methylmorphine-6-O-sulfate (14-O-MeM6SU) and examine its opioid properties (potency, affinity, efficacy) in receptor ligand binding and isolated tissues (mouse vas deferens, MVD and rat vas deferens, RVD bioassays). The results were then compared to the parent compounds morphine-6-O-sulfate (M6SU) and morphine, as well as the �- opioid receptor (MOR) selective agonist peptide [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). An additional objective was to compare the effect of subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) administered 14-O-MeM6SU, M6SU and morphine in thermal nociception, rat tail-flick (RTF) test. In MVD, the EC50 (nM) value was 4.38 for 14-O-MeM6SU, 102.81 for M6SU, 346.63 for morphine and 238.47 for DAMGO. The effect of 14-O-MeM6SU and DAMGO was antagonized by naloxone (NAL) with Ke value 1-2.00 nM. The Emax values (%) were 99.10, 36.87, 42.51 and 96.99 for 14-O-MeM6SU, M6SU, morphine and DAMGO, respectively. In RVD 14-O-MeM6SU and DAMGO but not M6SU or morphine showed agonist activity. In binding experiments the affinity of 14-OMeM6SU, M6SU, morphine and DAMGO for MOR was 1.12, 11.48, 4.37 and 3.24 nM, respectively. The selectivity of 14-O-MeM6SU was κ/µ= 269 and δ/µ= 9. In G-protein activation experiments, 14-O-MeM6SU and DAMGO showed higher Emax values than M6SU or morphine. S.c. or i.c.v-injected 14-O-MeM6SU, M6SU and morphine produced a dose and time-dependent increase in RTF response latency. 14-O-MeM6SU was the most potent. Our results showed that introduction of 14-O-Me in M6SU increased the binding affinity, agonist potency, and most importantly, the intrinsic efficacy (Emax).

Combined use of liquid chromatography with mass spectrometry and nuclear magnetic resonance for the identification of degradation compounds in an erythromycin formulation.

A commercial erythromycin formulation containing erythromycin A (EA) as the major compound showed the presence of an unknown degradation compound that was co-eluted with erythromycin E (EE) in the European Pharmacopoeia (Ph. Eur.) liquid chromatographic (LC) method. The amount of the degradation compound increased with respect to time. To separate this unknown (UNK1), investigation was performed with different LC methods coupled to ultraviolet detection (LC-UV). With the present Ph. Eur. method, the degradation compound could not be well separated. However, with the most selective LC-UV method (XTerra method), two more degradation products (UNK2 and UNK3) were found in the formulation which could not be observed using other methods because of their poor separation. By combining the results obtained with LC-UV, LC/MS and LC/NMR, the degradation products were identified as pseudoerythromycin A hemiketal (PsEAHK), erythromycin A enol ether carboxylic acid and erythromycin C enol ether carboxylic acid. PsEAHK is known to be a base-catalysed degradation product of EA, whereas the other two degradation products were newly identified.

Complementary fragmentation pattern analysis by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry confirmed the precious lignan content of Cirsium weeds.

In this paper, as novelties to the field, it is confirmed at first, that the fruits of Cirsium species, regarded as injurious weeds, do contain lignans, two, different butyrolactone-type glycoside/aglycone pairs: the well known arctiin/arctigenin and the particularly rare tracheloside/trachelogenin species. These experiences were supported by gas chromatography-mass spectrometry (GC-MS), by liquid chromatography tandem mass spectrometry (LC-MS/(MS)) and by nuclear magnetic resonance (NMR) spectroscopy. The study reflects the powerful impact of the complementary chromatographic mass fragmentation evidences resulting in the identification and quantification, the extremely rare, with on line technique not yet identified and described, tracheloside/trachelogenin pair lignans, without authentic standard compounds. Fragmentation pattern analysis of the trimethylsilyl (TMS) derivative of trachelogenin, based on GC-MS, via two different fragmentation pathways confirmed the detailed structure of the trachelogenin molecule. The complementary chromatographic evidences have been unambiguously confirmed, by (1)H and (13)C NMR analysis of trachelogenin, isolated by preparative chromatography. Identification and quantification of the fruit extracts of four Cirsium (C.) species (C. arvense, C. canum, C. oleraceum, and C. palustre), revealed that (i) all four species do accumulate the tracheloside/trachelogenin or the arctiin/arctigenin butyrolactone-type glycoside/aglycone pairs, (ii) the overwhelming part of lignans are present as glycosides (tracheloside 9.1-14.5 mg/g, arctiin 28.6-39.3 mg/g, expressed on dry fruit basis), (iii) their acidic and enzymatic hydrolyses to the corresponding aglycones, to trachelogenin and arctigenin are fast and quantitative and (iv) the many-sided beneficial trachelogenin and arctigenin can be prepared separately, without impurities, excellent for medicinal purposes.

Structural and physicochemical profiling of morphine and related compounds of therapeutic interest.

A concise account of the physicochemical properties of morphine and its derivatives of therapeutic interest is provided. Such properties include macroscopic and microscopic acid/base parameters, lipophilicity, solubility, permeability that all influence the fate of drugs in the body. The dependence of these parameters on pH is discussed and subsequent implications in drug administration and formulation are presented.

Methods for syntheses of N-methyl-DL-aspartic acid derivatives.

A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the alpha-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-alpha-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-alpha-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.

Site-specific acid-base properties of pholcodine and related compounds.

The acid-base properties of pholcodine, a cough-depressant agent, and related compounds including metabolites were studied by 1H NMR-pH titrations, and are characterised in terms of macroscopic and microscopic protonation constants. New N-methylated derivatives were also synthesized in order to quantitate site- and nucleus-specific protonation shifts and to unravel microscopic acid-base equilibria. The piperidine nitrogen was found to be 38 and 400 times more basic than its morpholine counterpart in pholcodine and norpholcodine, respectively. The protonation data show that the molecule of pholcodine bears an average of positive charge of 1.07 at physiological pH, preventing it from entering the central nervous system, a plausible reason for its lack of analgesic or addictive properties. The protonation constants of pholcodine and its derivatives are interpreted by comparing with related molecules of pharmaceutical interest. The pH-dependent relative concentrations of the variously protonated forms of pholcodine and morphine are depicted in distribution diagrams.

Enhancing effect of zinc on astroglial and cerebral endothelial histamine uptake.

We have studied the effect of zinc ion on the uptake of histamine (HA) into cultured astroglial and cerebral endothelial cells and established that Zn(2+) enhances the uptake of the amine dose-dependently and in remarkable extents by increasing the V(max) to about 3-fold (from 3.25 +/- 0.42 to 8.50 +/- 0.97 pmol/mg protein/min in astroglial cells) without altering the K(M) (0.20 +/- 0.03 microM) significantly. The stimulatory effect of zinc ion showed strong sensitivity for VUF 8407, an inhibitory compound of astroglial and cerebral endothelial uptake of HA. In the presence of 20 microM VUF 8407 the zinc-enhanced uptake was reduced by about 50% in both cell types. Binding measurements revealed increased capacities of the zinc-exposed HA binding (B(max)= 0.41 +/- 0.05 increased to 1.21 +/- 0.16 pmol/mg protein in astroglial membranes and B(max) = 0.25 +/- 0.03 enhanced to 1.05 +/- 0.12 pmol/mg protein in cerebral endothelial membranes) but statistically unchanged affinity of the ligand for HA carrier (K(D) values calculated as 35.2 +/- 3.4 nM and 45.1 +/- 3.8 nM for astroglial bindings; whereas 25 +/- 2.1 nM and 30 +/- 2.6 nM for cerebral endothelial bindings of the amine). The compound VUF 8407 reduced the B(max) of zinc-exposed HA binding of astroglial membranes but did not modify the K(D) of the zinc-exposed membrane significantly. The ex vivo experiments confirmed our in vitro findings; an i.c.v. dose of 0.4 micromol/kg ZnSO(4,) 24 hr after the injection, enhanced the uptake of [(3)H]HA into dissociated hypothalamic and cerebellar cells to about 2- and 3-fold, respectively. Present data clearly showed that zinc exposures enhance the astroglial and the cerebral endothelial uptake of HA in vitro and it might be considered that zinc produces similar effects in vivo. Free zinc may participate in the regulation of the extraneuronal HA concentration and this metal ion (endogenous or exogenous) might be favored in the removal of the amine from the interstitial space especially in conditions with relatively high HA.

[Use of crystal conversion reactions in the synthesis of pentacyclic alkaloid analogues]. / Kristály-átalakulási reakciók felhasználása pentaciklusos alkaloid analógok szintézisében.

During investigation of substitution of debenzorutaecarpine derivatives thermal cyclization reactions was observed to produce new fused heterocyclic systems. The optimal temperature of cyclizations was determined by differential calorimetric measurements of crystals of intermediate products. The thermolysis of 2-azido-3-formil-debenzorutaecarpine gave, after elimination nitrogen, izooxazolo-debenzorutaecarpine in good yield. The cyclization of the 2-azido-3-nitroso-debenzorutaecarpine readily led to oxadiazolo E-ring analogue of rutaecarpine. The thermal cyclization of 2-hydroxy-debenzorutaecarpines gave 2-oxo- and 4-oxo-pyrano E-ring analogues of rutaecarpine. The structure of substances was confirmed by spectroscopic methods. The synthesised compounds are considered as first representatives of new heterocyclic ring systems.

[Synthesis of carboline alkaloid analogues]. / Karbolinvázas alkaloid analógok szintézise.

Hybrid compounds were synthesized combining the structural features of two isomer natural indolalkaloids rutaecarpine (1) and nauclefine (2). These aza-bioisosteric analogues are the first representatives of a new heterocyclic ring system. Two alternative reaction routes were developed for the synthesis of pentacyclic compounds (4, 5) in which the key step is the Fischer indolization of the 6-phenylhydrazono-dipyrido[1,2-a;4,3-d]pirimidine-11-ones. In the case of E-ring substituted derivatives the synthesis was carried out via preparation and chemical transformation of pyrido[1,2-a]pirimidine-4-ones (14, 15) to 2-substituted-3-aza-rutaecarpines (17-20). Finally, the nucleophilic displacement of the chlorine atom of 2-chloro-3-aza-rutaecarpine (18) by dialkylaminoethylamine provided the 2-amino-substituted derivative (20) having improved physico-chemical properties and increased antitumour activity. The new compounds are characterized by UV, IR, 1H, 13C NMR spectroscopy.

Deconvolution of composite chromatographic peaks by simultaneous dual detections.

Composite chromatographic peaks are deconvoluted by a method that uses ratio formation from signals of simultaneous double detection. The method is generally suitable if two detector signals can simultaneously be acquired and their uses do not need any a priori assumption or mathematical shape analysis. A simple deduction makes the compound- and detector-specific intensive parameters explicit, which allows for the digital construction of directly invisible component peaks. The simultaneous double detection is shown to be superior to coupled detectors, sequentially fixed chromatograms, and subsequently synchronized peaks. The combination of circular dichroism and ultraviolet (UV) detection is shown to be especially advantageous in the analysis of enantiomers for which the other ratio-forming peak-deconvolution techniques have inherently been insensitive. The double chiroptical UV detection can be of further use to decompose overlapping peaks of nonenantiomeric compounds that are highly similar. The capacity of the method is exemplified by the analysis of morphine alkaloids, steroid oximes, and synthetic heterocycles.

Capillary electrophoresis separation of vinpocetine and related compounds: prediction of electrophoretic mobilities in partly aqueous media.

Offord's equation, a relationship between electrophoretic mobility and charge, size and shape of peptides, has been extended to quantitate the electrophoretic mobility of vinca alkaloids. Partly aqueous protonation constants and the derived theoretical mobilities have been proven to be able to predict experimental electrophoretic mobilities. In practice, seven vincamine derivatives of very low water-solubility were separated by capillary electrophoresis. Buffer total concentration, apparent pH and methanol content, the three most important parameters of the running buffer, were used in triangular resolution mapping to characterize separation. Even though electrophoresis is well known to slow down in partly aqueous media, under our optimized circumstances a baseline separation was achieved within 8 min in each case.

Population, acid-base, and redox properties of N-acetylcysteine conformers.

Rotamers of N-acetyl-L-cysteine (NAC, the most popular mucolytic drug) are characterized in terms of populations, site- and conformer-specific acid-base properties, reducing strength, and molecular pharmacology. A new, general relationship between the bulk- and rotamer-specific basicities is introduced. NAC at high pH predominantly exists in a trans thiolate-carboxylate rotameric form, whereas protonation promotes the occurrence of intramolecular hydrogen bond-forming isomers. Distribution curves of the rotamers are depicted as a function of pH. Rotamer-dependent thiolate basicities differ by up to 0.5 log k units. Carboxylate basicities show slight conformation-dependence only. The membrane-penetrating capabilities from various compartments of the body are assessed on the basis of the pH-dependent charge of the molecule. The thiol-disulfide half-cell potential is calculated, using the correlation between the thiolate basicity and oxidizability. The oxidation-reduction properties of NAC are compared to those of other biological thiols in their definite microscopic forms. The pharmacokinetic behavior is interpreted in terms of the physicochemical parameters, providing molecular/submolecular explanation for several therapeutic properties of NAC.

[7-aza analogs of quinazolino-carboline alkaloids. Synthesis, structure, characterization]. / Kinazolino-karbolinvázas alkaloidok 7-aza-analóg származékai szintézis, szerkezet, jellemzés.

The synthesis, structure and characterization of 8-substituted-7-azarutaecarpines (2) is described. These compounds were prepared by Fischer indolization of 3-amino-2-(1-phenylhydrazonoethyl)-4(3H)-quinazolin-one (5), followed by cyclocondensation with a series of aliphatic or aromatic aldehydes and formic acid or a Vilsmeyer-Haack reagent. The stereochemistry of compounds (2) was investigated by 1H nmr spectroscopy. It was found that the 8-substituents assume a quasi-axial position on the flattened boat conformation of ring C of (2), with the exception of ortho substituted phenyl groups, which occupy quasi-equatorial position. Semiempirical MO calculations support these conformational preferences.

[Synthesis and investigation of protonation properties and lipophilicity of some new quinazolone-3-carboxylic acid derivatives]. / Kinazolon-3-karbonsav származékok szintézise, protonálódási és lipofilitási vizsgálata.

Several new quinazolone-carboxylic acid derivatives as potential NMDA and AMPA receptor antagonists have been synthesized, and the protonation properties and lipophilicity of some representative molecules have also been studied. The protonation macroconstants (logK) of 4(3H)-quinazolone (Q0) and two 2-methyl-4-oxo-3H-quinazoline-3-carboxylic acids (Q1, Q2) were determined by pH-potentiometry. The acid-base chemistry of Q1 and Q2, where protein-bindings take place in an overlapping fashion, was described in terms of protonation microconstants (logk) as well. Microspeciation was carried out by UV-pH titration and deductive method. Microspeciation revealed remarkable differences between the two homologue compounds (Q1 and Q2), namely insertion of a second methylene moiety into the aliphatic acid side-chain reversed the predominantly zwitterion-involved protonation pathway into neutral form-involved one. Lipophilicity of our molecules was described by the octanol-water partition coefficients. The apparent partition coefficients of Q1 and Q2 were determined by shake-flask method and converted into true logP values using the protonation microconstants. The unexpected differences between their true logP values were explained, similarly to the different protonation pathways with conformational preferences and formation of intramolecular interactions. Out of the other 15 monoprotic quinazolone compounds the lipophilicity of 10 molecules (Q8-Q17, experimental set) was determined by RP-TLC method with the help of a calibration set consisting of 12 standard molecules, five quinazolones (Q3-Q7, determined by shake-flask method) and seven pyrido[1,2a]pyrimidines (PP1-PP7). The obtained logP values proved mostly the expected structure-property relationships. These physico-chemical investigations are pieces of predictive information for the pharmacokinetics of our compounds. These are also discussed in the paper.

Proton speciation and microspeciation of vinpocetine and related compounds in aqueous and biomimetic media.

PURPOSE: The determination of protonation macroconstants of twelve compounds in the vincamine drug family and the determination of protonation microconstants of cis- and trans-apovincaminic acid in media of various solvent composition to characterise their site-specific basicity and to estimate the concentration of the membrane-penetrating and receptor-binding forms. METHODS: UV-pH titrations have been used to determine the protonation macroconstants in 10-43 wt% methanol/water mixtures. Yasuda-Shedlovsky extrapolation was applied to obtain aqueous logK values for compounds sparingly soluble in water. Protonation microconstants were also determined by deductive methods for compounds of free carboxylic group. RESULTS: In the case of the two water-soluble compounds the extrapolated and the directly measured aqueous logK values were in good agreement, verifying all other extrapolated data. Compounds of cis-D/E ring anellation are 0.4-0.8 logK units more basic than their epimeric, trans counterparts. The pH-dependent distribution of apovin-caminic acid microspecies in aqueous and membrane-like media is depicted in microspeciation diagrams. CONCLUSIONS: The N(4) nitrogen is more shielded by the adjacent ethyl group in trans-D/E ring anellation eburnanes than in cis ones, as reflected by the protonation constants. Solvent-dependent basicity data predict superiority of trans isomers in lipophilicity and membrane-penetrating ability.

[Stereochemical aspects of narcotic and psychotropic drug action]. / A kábítószerhatás szterokémiai kérdései.

Medicinal drugs are predominantly, whereas narcotic and psychotropic drugs are exclusively exogenous compounds. Three further fundamental properties of the narcotic/psychotropic compounds of significant abuse potential is that they all are of natural (plant or microbial) origin, they contain a large number of chiral atoms, and they influence the neurotransmission processes in the central nervous system. For some of them, the existence of corresponding endogenous ligands have recently been reported. Since exogenous compounds and their endogenous ligands are assumed to bind the same target moiety of the receptor, several fundamental questions arise: To what extent can stereochemical relationships be established between the exogenous compound and its endogenous counterpart? Do they have superimposable moieties? Are the corresponding chiral atoms of the same configuration? Is there a chiral-genetic relationship between the exogenous and endogenous compound? Theoretical aspects and answers to all these questions are sought for morphine, cocaine, LSD, tetrahydrocannabinol, amphetamine and related molecules.

[Characterization of drug, narcotic and psychotropic drug chirality by statistical methods]. / Gyógyszer- és kábítószervegyületek kiralitásának statisztikai jellemzése.

The percentage of chiral entities among drug, narcotic drug and psychotropic compounds is steadily increasing. Receptors of the human body recognize the enantiomeric forms of constitutionally identical compounds as entirely different chemical agents. Based upon these facts, this paper reports the percentage of chiral compounds in the various pharmacological classes, and related data. Pertinent terms, such as eutomer, distomer, eudismic index, eudismic affinity quotient are defined. Differences in biological activity between eutomers and distomers are exemplified. The pharmacological classes and subclasses of highest chirality, and the "most chiral" active principles are shown. Some puzzling observations on pharmacological behaviour of stereoisomers are highlighted. The necessity of "racemate switch" in the pharmaceutical industry, and the significance of stereo-specific interactions between the drug, narcotic drug and psychotropic ligands, and complementary, "pocket" moieties of the human body are emphasized. Some features of enantiopharmacology, a fledgling science in the interface of stereochemistry and traditional pharmacology are introduced. The statistical treatment of asymmetric compounds in pharmacological classes and subclasses shows that presently, the percentage of chirality in drug categories is more characteristic of the origin of the compound than its target molecule.

[Microequilibrium of drug molecules]. / Gyógyszervegyületek mikroegyensúlyai.

Protonation macro-, micro-, and submicro constants are physico-chemical parameters of crucial importance, concerning the fate of bio-, drug, and narcotic drug molecules in the body and in protic solvents. The most important characteristics, relationships, applications and biological significance of these parameters are reviewed, using acetylcysteine and cysteine, as examples. The mucolytic effect of acetylcysteine, an active principle in numerous drugs, is interpreted in terms of protonation state of the molecule and its thiolate site. Microscopic protonation constants of acetylcysteine, data that have not previously appeared, are also reported.

[Preparation and characterization of potential antineoplastic agents]. / Potenciális daganatellenes hatású vegyületek elóállítása és vizsgálata.

A parallel combinatorial library of over sixteen hundred compounds has been designed and synthesized for the development of new potential peptidomimetic protein tyrosine kinase (PTK) inhibitor leads that is aimed for intervening with the substrate binding site of the pp60c-src enzyme. The new structures were based on known PTK inhibitors having at least two variously substituted aromatic moieties attached by spacer groups of different length and flexibility. Eleven bis-aryl type inhibitory compounds were found in the range of 18-100 micromolar IC50 concentrations from combinations of twelve different substituents. Molecular modeling of the active compounds showed a characteristic distance of 13-14 A between the farthest sp2 carbon atoms of the two aromatic rings. Conformational analysis of several peptide substrates recently found for pp60c-src PTK [5,6,7] showed that the energy minimized conformers had the same distance between two aromatic moieties. Several compounds in the library not only showed remarkable PTK inhibitory activity but also a significant apoptosis inducing effect on HT-29 human colon tumor cells.