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Non-quaternary and N-benzyl quaternary chiral quinuclidinium esters1, 2 and amides3, 4 of pivalic, butyric and benzoic acid were evaluated as substrates and/or inhibitors of cholinesterases. Inhibitory effect of quinuclidinium amides on acetylcholinesterase (AChE from electric eel) and butyrylcholinesterase (BuChE form horse serum) were determined using a spectrophotometric method (Ellman’ s reagent) in a microplate assay.5 Hydrolysis kinetics of chiral esters (HPLC analysis, substrate: benzoylcholine) revealed that (R)-enantiomers of tested esters are much better substrates than (S)- enantiomers. On the other hand, (S)-enantiomers of esters and amides showed higher affinity toward ChEs except in the case of the butyric acid derivatives. In order to explain experimental data concerning activity of enantiomers, molecular docking simulations were performed using AutoDock 4.0 suite of programs1. We employed flexible ligand docking (Lamarckian Genetic Algorithm (LGA) search method) allowing all possible torsions in molecules. The results are compared with those for benzoyl choline and galanthamine. It was shown that enantiomeric preference of cholinesterases, as well as rates of hydrolysis, are governed primarily by electrostatic interactions and steric limitations and in the choline subsite. The differences in orientation and relative energies will be presented and discussed.

Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH3, Py-4-Br, Py-4-Cl and Py-4-NO2) were previously evaluated as inhibitors of human erythrocyte acetylcholinesterase (AChE ; EC 3.1.1.7) as well as reactivators of tabun-inhibited AChE (Z. Kovarik et al.). Therefore, we performed docking studies to elucidate the differences in oximes potency. The orientations of all studied oximes in the active site of human AChE have been proposed by flexible ligand docking with AutoDock 3.0. Two forms of each oxime - protonated (=NOH) and deprotonated (=NO-) oxime group - were used for docking simulations. Analyses of the obtained complexes revealed the presence of numerous hydrogen bonds and close contacts between the oximes and the residues in the active site. It can be seen that ligands are oriented in such a way that the hydrogen bond is always formed between the oxime oxygen and various amino acids. Furthermore aromatic rings of the ligand are stabilized via the  - interactions mainly with Trp86, occupying in the most cases the choline binding site. Some clusters of geometries are found to be positioned toward the rim of the active site gorge making H-bonds with the Thr75 and Ser293 and  - interactions with some aromatic amino acids present in the active site gorge. The main difference in binding of Py-4-Br and Py-4-NO2 is in that region ; in the case of Py-4-Br there are much more clusters of geometries which is probably due to the size of that ligand. For Py-4-H none of the clusters in that region were found. Final docked energies predicted correctly the relative order of the inhibition potency of compounds (experimentally: Py-4-Br < Py-4-Cl < Py-4-CH3 < Py-4-H < Py-4-NO2) except in the case of Py-4-CH3 which was determined as a second strongest inhibitor. For the best reactivator of tabun-inhibited AChE, Py-4-Br, the most probable orientation which can result in an attack on the phosphorus atom of the tabun-phosphorylated human AChE was proposed.

It is known that chiral 1, 1`-bis-2-naphthols are useful agents for the NMR determination of enantiomeric purity of some amino alcohols and other compounds [1]. Therefore, we investigated the potential of (R)-1, 1-bis-2-naphthol (RBN) as a chiral solvating agent in the case of prepared tertiary and quaternary quinuclidine esters. We have prepared racemates and enantiomerically pure esters of acetic, pivalic, butyric and benzoic acid as well as their quaternary N-methyl and N-benzyl derivatives. Optical rotation measurements and 1H NMR using RBN as a chiral solvating agent were used to check optical purities of the prepared compounds. Spectra were recorded with a Gemini 300 MHz NMR spectrometer, using CDCl3 as a solvent and adding 5 molar equivalents of RBN. A significant shift of all quinuclidinium protons toward the higher field was observed for all compounds, indicating that these protons are under the influence of the ring current effect of the binaphthyl system. The splitting of the quinuclidinium proton resonances into two signals was more significant in the case of the racemic quaternary derivatives and the magnitude of the non-equivalence was greatest for the splitting of the quinuclidinium 4-H resonance of the quaternary benzoates [2]. Among the other tested esters the splitting of the 4-H was only observed in the N-benzyl derivatives but magnitude of the non-equivalence between enantiomers was much lower indicating the importance of the p-p interactions among the ester and RBN. Proton resonances of 4-H were assigned by the use of 2D 1H COSY spectra. From the NMR spectra of each enantiomer it was determined that the 4-H signal at a higher field belongs to the (S)-enantiomer. [1] F. Toda et al., Bull. Chem. Soc. Jpn. 61 (1988) 4167. [2] I. Primožić et al., Eur. J. Org. Chem. (2003) 295.

Many esters of optically active quinuclidin-3-ol have been tested and recognized as pharamacological agents. Previous work also showed that butyryl cholinesterase (BChE ; 3.1.1.8.) catalyzed the hydrolysis of N-benzyl protected quinuclidinium esters in a stereoselective manner. In order to study the influence of substrate structure on kinetic resolution, racemic, (R)- and (S)-esters of quinuclidin-3-ol and isobutyric acid were synthesized as well as their racemic and chiral, quaternary N-benzyl and N-p-bromobenzyl derivatives. Racemic quinuclidin-3-yl acetate was synthesized in the first step by esterification of quinuclidin-3-ol with acetic anhydride. Resolution of the racemate with L- and D-tartaric acid, enantiomerically pure acetates were obtained. Chiral (R)- and (S)-quinuclidin-3-ols were prepared by hydrolysis of (R)- and (S)-acetates, respectively. N-Benzyl and N-p-bromobenzyl derivatives were synthesized in the next step by quaternization of appropriate chiral or racemic esters with benzyl bromide and p-bromobenzyl bromide. The rates of hydrolysis were monitored (HPLC) at 30°C in 0.1 M phosphate buffer, pH 7.4. Kinetic studies revealed that (R)-enatiomers of the tested esters are much better substrates than (S)-enantiomers. Introduction of the bromine atom in the para position of the phenyl ring resulted in a remarkable improvement of hydrolysis enantioselectivity.

The stereoselectivity of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) was studied in the interaction with enantiomers of quinuclidin-3-yl acetates as substrates as well as in the reversible inhibition of the enzymes by enantiomers of quinuclidin-3-ol derivatives. For that purpose (R)- and (S)-alcohols and their acetates as well as their quaternary N-methyl and N-benzyl derivatives were prepared and characterized.

Različitim mikroskopskim metodama istražen je utjecaj duljine poveznika na asocijaciju u otopini te termička svojstva u krutom stanju dimernih površinski aktivnih tvari.

This report summarizes studies on pyridinium, imidazolium, and quinuclidinium oximes, which were prepared in laboratories in Croatia since the middle of the 1970s. The prepared compounds, 176 in all, include 157 new compounds. The majority of prepared compounds are monoquaternary and bisquaternary pyridinium oximes. In vitro studies comprised reversible inhibition of cholinesterases, protection of cholinesterases from phosphylation by organophosphorus compounds (OP), and reactivation of the phosphylated enzyme. In vivo studies of the oximes as antidotes in experimental poisoning by OP compounds are also reviewed. The OP compounds were predominantly chemical warfare (CW) nerve agents but also included some related pesticides.

Acetilkolinesteraza (acetikolin-acetilhidrolaza, E. C. 3.1.1.7) enzim je kojeg inhibiraju neki organofosforni spojevi među koje spadaju i oni koji se koriste kao pesticidi te živčani bojni otrovi. Iako su biskvateni piridinijevi oksimi dobro poznati kao antidoti, univerzalni antidot koji bi bio udovoljavajuće djelotvoran pri trovanju organofosfornim spojevima još uvijek nije pronađen. U ovom preglednom članku opisane su sinteze i antidotska svojstva dviju klasa spojeva- imidazolijevih i kinuklidinijevih-obje sa mogućim višestrukim učinkom u kolinergičnom sustavu. N- alkilimidazolijevi aldoksimski derivati spojevi su koji pokazuju visoki afinitete za katalitičko mjesto enzima. Primarni antidotski učinak imidazolijevih oksima je reaktivacija fosforilirane AChE. Antidotska djelotvornost 3- supstituiranih kinuklidinijevih derivata rezultata je njihove interakcije s AChE, ali i s drugim receptorima u kolinergičkom sustavu (npr. supresija presinaptičke sinteze acetilkolina). Do sada je sintetizirani nekoliko serija biskvaternih imidazolio-piridinijevih, piridinio- kinuklidinijevih te imidazolio-kinuklidinijevih oksima kako bi se istražila svojstva spojeva koji sadrže u svojoj strukturi po dvije aktivne funkcije. Neki su se od tih derivata pokazali dobrim antidotima organofosfornih spojeva. Raspravljena su svojstva pripravljenioh spojeva kao reverzibilnih i progresivnih inhibitora acetilkolinesteraze, acilirajućeg agensa kao i njihova sposobnost zaštite enzima od fosforiliranja te mogućnosti reaktivacije AChE fosforilirane organofosfornim spojevima.

Quaternary N-benzyl (R)- and (S)-esters of quinuclidin-3-ol and amides of quinuclidin-3-yl amine and pivalic, butyric and benzoic acid were synthesized. Kinetic studies revealed that (R)-enantiomers of tested esters are much better substrates than (S)-enantiomers. On the other hand, (S)-enantiomers of esters and amides showed higher affinity toward BChE except in the case of the butyric acid derivatives. In order to explain experimental data concerning reactivity of enantiomers, quantum chemical calculations (ONIOM scheme, B3LYP/3-21G ; PM3) of the stable species in the acylation step were performed. It was shown that enantiomeric preference of BChE, as well as rates of hydrolysis, are governed in large part by steric limitations and electrostatic interactions in the choline subsite.

The (R)- and (S)- enantiomers of quinuclidin-3-ol, (R)- and (S)-QOH were prepared by resolution of commercially available racemic quinuclidin-3-ol using D- and L- tartaric acid. (R)- and (S)- quinuclidin-3-yl-acetates, (R)- and (S)-QA were synthesized by esterification of (R)- and (S)-QOH with acetic anhydride. Subsequently both enantiomers of quaternary N-metyl alcohols and acetates (MetQOH and MetQA) were prepared using methyl iodide as a quaternization agent. Enantiomers of quaternary N-benzyl alcohols and acetates (BzQOH and BzQA) were synthesized in a similar manner by using benzyl bromid as a quaternization agent. All prepared compounds were then tested as substrates of acetylcholinesterase and butyrylcholinesterase, and/or inhibitors of the cholinesterases.

Pripravljena su četiri kvaterna estera kinuklidin- 3-ola: esteri octene, pivalinske, maslačne i benzojeve kiseline. Resolucijom uz pomoć D- i L- vinske kiseline [1] pripravljeni su enantiomerno čisti (R)- i (S)-kinuklidin-3-ol. Esterifikacijom s odgovarajućim anhidridom te zatim kvaternizacijom s benzil-bromidom pripravljeni su kiralni kvaterni N-benzilni esteri. Pripravljeni esteri poslužili su kao supstrati za butirilkolinesterazu. Ispitana je aktivnost te enantioselektivnost ovog enzima u reakcijama hidrolize navedenih estera u vodenom mediju. Uspoređene su brzine reakcija (S)- i (R)- enantiomera (4 mM koncentracija supstrata) te brzine hidrolize 2 mM otopine (R)-estera i 4 mM racemata. Pokazalo se da su esteri maslačne kiseline najbolji supstrati za enzim te da su (R)- enantiomeri puno bolji supstrati za enzim od (S)- enantiomera. Među (R)-esterima acetat je hidroliziran 7×, pivalat 8×, a benzoat 9× sporije nego (R)-ester maslačne kiseline. Još veća selektivnost pokazala se u slučaju (S)- enantiomera: acetat je hidroliziran 10×, benzoat 32×, a pivalat 103× sporije nego (S)-ester maslačne kiseline. Usporedbom brzina odgovarajućih enantiomera izračunato je da su reakcije brže za 320, 450, 1180 i 4360 puta za (R)-enantiomere estera maslačne, octene, benzojeve odnosno pivalinske kiseline. Nadalje, utvđeno je da (S)- enantiomeri inhibiraju enzim. Najveća razlika u brzini opažena je pri hidrolizi racemičnog estera benzojeve kiseline: hidroliza se odvijala 5× sporije od hidrolize (R)-enantiomera iste koncentracije. [1] B. Ringdahl, B. Resul, R. Dahlbom, Acta Pharm. Suec. 16 (1979) 281-283.

Optically active quinuclidin-3-ol is an important intermediate in the preparation of physiologically or pharmacologically active compounds. Thus, new methods for the efficient production of chiral quinuclidin-3-ols are still examined. It has been shown that stereoselectivity of hydrolyses catalysed by butyrylcholinesterase (BChE ; EC 3.1.1.8) was in favour of the (R)-enantiomers in the case of quinuclidinium benzoates.1-3 In this study racemic, (R)- and (S)- esters of quinuclidin-3-ol and propionic acid were synthesized as well as their racemic and chiral, quaternary N-benzyl and N-p-brombenzyl derivatives in order to investigate their hydrolysis by horse serum BChE. The rates of hydrolyses were monitored (HPLC) at 30 oC in 0.1 M phosphate buffer pH 7.4 (2 mM concentrations of chiral esters). Kinetic study revealed that (R)-enantiomers of tested esters are much better substrates than (S)-enantiomers. In the case of N-benzyl protected esters, the rate of hydrolysis of (R)-ester was 490 fold faster than that of (S)-ester. Introduction of bromine atom in the para position on the phenyl ring of benzyl moiety resulted in a remarkable improvement in the enantioselectivity of hydrolysis: under the same experimental conditions it was not possible to monitor the very slow reaction of (S)-N-p-brombenzyl ester. From that data it can be concluded that BChE is suitable as a biocatalyst for the resolution of racemic, N-benzyl protected quinuclidinium esters. 1. Ines Primozic, Tomica Hrenar, Srdjanka Tomic and Zlatko Meic, Journal of Physical Organic Chemistry 15 (2002) 608-614. 2. Ines Primozic, Tomica Hrenar, Srdjanka Tomic and Zlatko Meic, European Journal of Organic Chemistry (2003) 295-301. 3. Ines Primozic, Tomica Hrenar, Srdjanka Tomic and Zlatko Meic, Croatica Chemica Acta 76 (2003) 93-99.

(&plusmn ; )-, (R)- and (S)- esters of quinuclidin-3-ol and acetic, pivalic, butyric and benzoic acid were synthesized as well as their racemic and chiral, quaternary N-benzyl derivatives. Butyrylcholinesterase (BChE) was identified, among other tested esterases, as a catalyst of choice in hydrolysis reactions of prepared quaternary esters. Kinetic studies revealed that (R)-enantiomers of tested esters are much better substrates than (S)-enantiomers. On the other hand, (S)-enantiomers showed higher affinity toward BChE (3-60 micro M). In order to explain experimental data concerning reactivity of enantiomers, quantum chemical calculations (ONIOM scheme, B3LYP/3-21G:PM3) of the stable species in the acylation step were performed. It was shown that enantiomeric preference of BChE, as well as rates of hydrolysis, are governed in large part by steric limitations and electrostatic interactions in the choline subsite. Optimal positioning of the enantiomers in the active site resulted in a different twisted structures of quinuclidinium skeleton (Figure 1) and obvious steric hindrance for (S)-esters, explaining why they were a poor substrates for the enzyme.

In order to investigate the dependance of enzyme activity and enantioselectivity on the ester's quaternary amonium group, three diferent N-quaternary quinuclidinol propanoates were prepared. To prepare chiral esters of quinuclidin-3-ol and propanoic acid, first, racemic quinuclidin-3-yl acetate was synthesized by esterification of quinuclidin-3- ol with acetic anhydride. Resolution of the racemate with L- and D-tartaric acid, enantiomericaly pure acetates were obtained. Chiral (R)- and (S)-quinuclidin-3-ols were prepared by hydrolysis of (R)- and (S)-acetates respectively. N-benzyl derivatives were synthesized in the next step by quaternization of appropriate chiral or racemic ester with benzyl bromide, p-methylbenzyl bromide and p- nitrobenzyl bromide. The strucutures and purity of all compounds were deducted from IR, one- and two-dimensional NMR, and optical purity by optical rotation measurement. Butyrylcholinesterase (BChE) was identified, among other tested esterases, as a catalyst of choice in hydrolysis reactions of prepared quaternary esters. All compounds were then tested as substrates of serum butyrylcholinesterase (BChE ; EC 3.1.1.8). The initial rates of hydrolyses were monitored (HPLC) at 30°C in 0.1 M phosphate buffer pH=7.4 (2 mMconcentration of substrates). The preface of BChE towards (R)-enantiomers and unsubstitued N-benzyl derivatives as well as the difference in the rates of hydrolysis among enantiomers will be discussed.

Three quaternary, asymmetric, dimeric surfactants with two hydrocarbon chains differing in length (tetradecyl and dodecyl) and with different polymethylene spacers N, N, N&cent , N&cent -tetramethylethane (12-2-14), N, N, N&cent , N&cent -tetramethylehexane (12-6-14) and N, N, N&cent , N&cent - tetramethyldecane (12-10-14) were prepared. Their solid phase and thermal behavior were examined by X-ray diffraction, spectral, thermal and optical microscopic studies. Structure of 12-2-14 contains interdigitated alkyl chain layers, 12-6-14 monolayers of molecules with tilted alkyl chains, while 12-10-14 exhibits liquid crystalline structure even at room temperature. Molecules with a shorter spacer are all in cis-configuration, while those with longer ones are all in trans-configuration. On heating all compounds exhibited the conformational disordering of the alkyl chain during phase transitions in the solid state at lower temperatures and the bidimensional disordering of the ionic layers during the solid crystalline to liquid crystalline transitions. Phase transitions in the solid state include structural varieties, polymorphs and smectic liquid crystals. Crystal structure, number of phase transitions and thermodynamic parameters strongly depend on the spacer length ; i.e., on the global configuration of molecules. Temperatures of the first solid state, solid crystal &#8211 liquid crystal and liquid crystal &#8211 isotropic melt transitions exhibit maximum for s = 6. Polarizing microscopy revealed characteristic textures of the smectic phases in the case of 12-2-14 and 12-10-14.

Adsorption at interfaces, association in aqueous solutions, and thermal solid phase transitions of dissymmetric gemini surfactants were investigated by several methods.

Pripravljeni su (&plusmn ; )-, (R)- i (S)- esteri kinuklidin-3-ola te octene, pivalinske, maslačne i benzojeve kiseline kao i njihovi racemični i kiralni, kvaterni N-metilni i N-benzilni derivati. Optička čistoća spojeva određivana je polarimetrijski i snimanjem 1H NMR spektara uz 1, 1'-bi-2-naftol kao kiralni solvatirajući reagens. Među testiranim esterazama odabranim kao katalizatorima za hidrolizu pripravljenih estera, butirilkolinesteraza (BChE) je odabrana kao najpogodniji katalizator. Kinetika hidrolize katalizirane s BChE praćena je (HPLC) za sve estere benzojeve kiseline, sve N-benzilne derivate i benzoilkolin. Ustanovljeno je da su (R)-enantiomeri estera kinuklidin-3-ola znatno bolji supstrati od (S)-enantiomera koji su pokazali visoki afinitet prema enzimu. Iz dobivenih kinetičkih podataka proizlazi da je moguće uspješno izvesti resoluciju racemičnih N-benzilnih estera s BChE. Kombiniranim kvantno-kemijskim i semiempirijskim proračunima (ONIOM shema, B3LYP/3-21G:PM3) određene su strukture Michaelisovih kompleksa i tetraedarskih međuprodukata estera benzojeve kiseline unutar modela aktivnog mjesta BChE. Molekulskim uklapanjem utvrđeno je da postoji korelacija između izračunatih i eksperimentalno procijenjenih slobodnih energija vezanja.

A novel series of dissymmetric bisquarternary ammonium surfactants with a general formula C12H25(CH3)2N(CH2)sN(CH3)2C14H29Br2 (12-s-14), where s = 2, 6, 10 has been synthetised and characterised by FTIR, NMR and electrospray mass spectroscopy. Effect of the carbon number of the alkanediyl spacer on the adsorption and association in aqueous solution were studied by surface tension, conductometry, potentiometry, viscometry, dynamic light scattering and cryo-TEM. Surface and association properties show a behavior closely related to that of their symmetric counterparts. The length of the spacer chains, as well as the attractive and repulsive interactions of the surfactant molecules play an important role in determining the equilibrium area per molecule affecting both, the adsorption at the air/solution interface and morphology of aggregates formed in solution.

A series of dissymmetric gemini surfactants were prepared from n-dodecyldimethylammonium and n-tetradecyldimethylammonium bromide and linked by polymethylene chains (spacer), the number of carbon atoms in a spacer being 2, 6 and 10. Their solid phase properties were examined by X-ray diffraction, spectral, thermal and optical microscopic studies. The increase of the number of carbon atoms in the spacer changed the structure and thickness of bilayers. On heating, successive phase transitions (several solid crystalline-solid crystalline, solid crystalline-liquid crystalline and liquid crystalline-isotropic liquid) were observed. The number of polymorphs depended on the lengths of the spacer. Polarizing microscopy revealed a characteristic texture of the smectic phase. Crystallization from melted samples was kinetically controlled.

Four chiral, quaternary, N-methyl and N-benzyl derivatives of (R)- and (S)-quinuclidin-3-yl benzoates were synthesized and studied as substrates of horse serum butyrylcholinesterase (BChE). The kcat for the substrates decreased in order (R)-N-methyl > (R)-N-benzyl (2-fold slower) >>(S)-N-methyl (70-fold slower reaction), while (S)-N-benzyl ester acts as an inhibitor of the enzyme, KD= 3 &#956 ; ; ; M. The kinetic of inhibition indicated that binding to the catalytic site of BChE occured. The KM values of substrates revealed that the binding affinity of (R)-N-benzyl derivative toward BChE is higher than that of (S)-N-methyl ester. From the ratio of the enantiomeric kcat/KM values, an enantiomeric excess of 95 % was calculated for N-methyl derivatives. Thus, resolution of racemic N-methyl and N-benzyl quinuclidinium esters can be achieved by the hydrolysis catalyzed with BChE. The orientations of all studied benzoate esters in the active site of human BChE have been proposed by flexible ligand docking with AutoDock 3.0 suite of programs.[1] The main differences in orientations of obtained Michaelis complexes were found in the ammonium electrostatic region which include interactions of the ammonium moiety of substrates with the indole ring of Trp84 and carboxyl group of Glu199. [1] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K. Belew and A. J. Olson, J. Comput. Chem. 19 (1998) 1639-1662.

Chiral (S)-quinuclidin-3-yl benzoate (SQBz) and (R)-quinuclidin-3-yl benzoate (RQBz) were prepared and interactions with cholinesterase were studied in order to determine the usefulness of a biocatalytic method for resolution of racemic esters of quinuclidin-3-ol. RQBz and SQBz, as well as benzoylcholine chloride (BzCh), were tested as substrates for serum butyrylcholinesterase (BChE, EC 3.1.1.8). It was shown that nonquaternized S and RQBz are poorer substrates for BChE than BzCh. In case of RQBz the reaction rate was 7-fold, while for SQBz 100-fold slower than for tested choline ester. (R)-enantiomer of quinuclidinyl esters was shown 18-fold preference for BChE over (S)-enantiomer. Thus, molecular modelling studies were undertaken in order to understand the observed differences of tested compounds in binding to BChE, as well as to predict possible structural changes to further improve selectivities. Conformational spaces of SQBz and RQBz were analyzed at semiempirical level and three stable conformers were found for each enantiomer. Stable conformers were optimized with the DFT quantum-chemical method using B3LYP functional and 6-31G* basis set. Docking simulations were performed using AutoDock 3.0 suite of programs1. We employed two types of calculations: rigid-body docking of all optimized conformers and flexible ligand docking allowing all torsions in molecules. Lamarckian Genetic Algorithm (LGA) search method was used in both simulations. The results are compared with those for BzCh. The difference in orientation and relative energies will be presented and discussed.

In our previous study quinuclidinium compounds were shown to protect acetylcholinesterase against phosphorylation by soman and VX, and to protect mice against soman by increasing its LD50. In this study (R)- and (S)-enantiomers of N-benzyl-3-hydroxyquinuclidinium chloride (QOHBz) were prepared in order to test the difference in their affinity for cholinesterases. Chiral (R)- and (S)-quinuclidin-3-ols were prepared by resolution of racemic quinuclidin-3-il acetate with L- and D-tartaric acid followed by hydrolysis. (R)-N-benzyl-3-hydroxyquinuclidinium chloride (R-QOHBz) and (S)-N-benzyl-3-hydroxyquinuclidinium chloride (S-QOHBz) were synthesized by quaternization of the appropriate chiral alcohol with benzyl chloride. The structure and purity of all compounds were determined by MS, IR, one- and two-dimensional NMR, and optical purities by optical rotation measurement. R- and S-QOHBz were tested for inhibition of human erythrocyte acetylcholinesterase (AChE) and serum butyrylcholinesterase (BChE). Reversible inhibition of AChE or BChE by R- and S-QOHBz was measured with acetylthiocholine as substrate (0.1-1.0 mM). The dissociation constants of enzyme-inhibitor complexes were determined from Hunter-Downs plots. The dissociation constants of AChE were 0.26 and 0.67 mM for (S)- and (R)-enantiomers and those of BChE were 0.053 and 0.084 mM for (S)- and (R)-enantiomers respectively. R- and S-QOHBz have higher affinities for AChE and BChE than their N-methyl analogues.

Racemic quinuclidin-3-il acetate was synthesized by esterification of quinuclidin-3- ol with acetic anhydride. (R)- and (S)- quinuclidin-3-il acetates were obtained by resolution of the racemate with L- and D- tartaric acid. Chiral (R)- and (S)-quinuclidin- 3-ols were prepared by hydrolysis of (R)- and (S)-acetates. (R)-N-methyl-3-hydroxyqui- nuclidinium iodide (R-QOH) and (S)-N-methyl-3- hydroxyqui-nuclidinium iodide (S-QOH) were synthesized by quaternisation of appropriate chiral alcohol with methyl iodide. The structures and purity of all compounds were determined by IR, one- and two-dimensional NMR, and optical purity by optical rotation measurement. R- and S-QOH were then tested for inhibition of human erythrocyte acetylcholinesterase (AChE ; EC 3.1.1.7) and serum butyrylcholinesterase (BChE ; EC 3.1.1.8). Reversible inhibition of AChE or BChE by R- and S-QOH (1.0 - 10 mM) was measured at 37 oC in 0.1 M phosphate buffer pH=7.4 with acetylthiocholine as the substrate (0.1 to 5.0 mM). The dissociation constants of enzyme- inhibitor complexes were determined from Hunter-Downs plots. The dissociation constants for AChE and both enantiomers were the same (1.2 mM). The dissociation constants for BChE were 2.1 and 0.87 mM for R- and S-QOH respectively.

A novel asymmetric dimeric quaternary ammonium salts (gemini surfactants)have been synthesized. The composition, structure, morphology and thermal behavior of new compounds are studied. A theoretical model for the interfacial behavior of dimeric surfactants and its dependence on the spacer length was used to elucidate the morphologies of aggregates formed in the aqueous solution.

Istraživanja antidotske djelotvornosti pri trovanju organofosfornim spojevima heterocikličkih oksima modeliranih prema piridinijevim aldoksimima, odavno su proširena i na derivate imidazola. U nastavku naših istraživanja imidazolijevih aldoksima, pripravljeni su novi 1- vinil-imidazolijevi derivati: monokvaterni (1- metil), biskvaterni simetrični derivat povezan oksidimetilenskim lancem te dva biskvaterna imidazolio-kinuklidinijeva derivata povezana oksidimetilenskim, odnosno trimetilenskim lancem. Pripravljene supstancije karakterizirane su spektroskopskim metodama. UV spektrima i analizom povezivanja signala u NOESY spektrima studirana je molekulska struktura pripravljenih spojeva. Ab initio proračunima na HF razini teorije uz korištenje 3-21G osnovnog skupa proučavane su geometrije konformera pripravljenih spojeva s obzirom na rotaciju vinilne i aldoksimske skupine.

It is known that compounds which contain quinuclidinium and imidazolium subunits possess a wide range of biological activities in the cholinergic system. Some of them were shown to be antidotes against organophosphorus, as well. In this work we describe the synthesis of N-methyl and N-(3-(2-hydrxyiminomethyl-3-methyl-1- imidazolio)-2-oxapropyl derivatives of 3-hydroxy, 3-dimethylcarbamoyloxy and 3-acetyloxy quinuclidines. Molecular modeling studies were undertaken in order to gain some insight on how different enantiomers of synthesized compounds might interact with acetylcholinesterase (AChE) binding sites. Synthesized quinuclidinium derivatives have been studied as reversible inhibitors of AChE and protectors against phosphorylation of the enzyme by some warfare agents.

It is known that antidotal properties of 3- hydroxyquinuclidinium derivatives are the consequence of their interaction with acetycholinesterase (AChE) and/or other receptors in the cholinergic system (1). On the other hand, the primary antidotal effect of imidazolium oximes is reactivation of phosphorylated AChE. Several imidazolio-quinuclidinium oximes have been synthesized in order to investigate the properties of compounds containing both moieties in the same molecule. We describe the synthesis of N-(3-(2- hydroxyiminomethyl-3-methyl-1-imidazolio)propyl-3- oxoquinuclidinium diiodide (I) and N-(3-(2- hydroxyiminomethyl-3-methyl-1-imidazolio)-2- oxapropyl derivatives of 3-oxo (II), 3-hydroxy (III), 3-dimethylcarbamoyloxy (IV) and 3-acetyloxy (V) quinuclidines. Different substituents have been introduced in the 3-position of the quinuclidinium moiety in attempt to increase biological activity of new compounds. Furthermore, compound I was synthesized to evaluate the possible importance of oxygen instead of carbon in the chain connecting quinuclidinium and imidazolium parts. Synthesized compounds were studied for their mode of binding to AChE, their effect upon phosphorylation of AChE by Soman and VX and their effect upon Soman intoxicated mice (2).

Sintetiziran je niz kinuklidinijevih i imidazolijevih derivata radi izučavanja antidotskih svojstava spojeva koji sadrže 3-supstituirani kinuklidinijev, 3-supstituirani imidazolijev 2-aldoksimski te oba heterociklička prstena zajedno. Spojevi su identificirani spektroskopskim metodama (IR, 1D (-H, 13-C (APT)) i 2D NMR (h,H-COSY, NOESY i HECTOR).

In this paper, new synthesized oxime derivatives of imidazole and quinuclidines were tested in vitro using human erythrocyte AChE inhibited by soman and in vivo using soman poisoned mice. The inhibitory power (IC50, concentration of the test compounds, oximes, that inhibit to 50 % of the AChE activity), acute toxicity (LD 50 ), as well as reactivating and protective capacities, with respect to soman-inhibited AChE were tested for each of the synthesized oximes. Antidotal characteristics of the new syntesized oximes were compared and related to their chemical structures. Derivatives of imidazoles demonstrated mostly weak reactivating and protective characteristics, both for the in vitro and in vivo cases. These characteristics were independent of the substituents on N phenyl imidazolium oximes. Of the imidazoles, only BMR-3 oxime strongly reactivated the soman inhibited AChE (55%), i.e., in vitro, for human erythrocytes. BMR-4 oxime, in combination with atropine sulfate, provided effective protection in vivo (for mice) against 1.8 and 2.2 LD50 of soman. On the contrary, all tested quinuclidine oximes showed very good protection in vivo (for example, BM-1 protects against 4 x LD 50 of soman), but were ineffective in vitro, i.e., against soman-inhibited human erythrocyte AChE. The results indicate that in vivo effectiveness of quinuclidine oximes against soman poisoning is not telated to their reactivating or protective potentials for AChE measured in in vitro experiments ; their good protective effect is more likely to be related to other mechanisms of the cholinergic system.

3-Carbamoyloxy, 3-hydroxy and 3-oxo quinuclidinium derivatives were synthesized as possible antidotes of organophosphorus poisoning.

The in vitro activity of human erythrocyte acetylcholinesterase (AChE) and protective activity against soman dichlorovos, and propoxur poisoning in male mice (in vivo experiments) were determined by using bis- phenyl imidazolium (2B) and imidazole- quinuclidinium oximes (BM-1). Oxime 2B strongly inhibits the AChE, and has a good reacitvation potency, but weak protective ability, against AChE inhibited by these agents. Oxime BM-1 inhibits weakly AChE and its in vitro activity against inhibited AChE is negligible. In vivo, this oxime is a very good protective agent against inhibitors tested in this paper. BM-1, given with atropine sulphate, provided a 100 % protection in male mice gainsit 4 x LD 50 soman, 11 x LD 50 dichlorovos, and 16 x LD 50 propoxur, respecitvely. The results indicate that in vivo effectiveness of quinuclidinium oxime is not related to its reactivatin or protective potentials for AChe, as shown by the in vitro experiments. The in vivo action of this BM-1 oxime must be attributed to mechanism not yet well defined.

Three compounds: N-methyl 3-oxo quinuclidinium iodide (I), 2-hydroxyiminomethyl 1, 3-dimethylimidazolium iodide (II) and this two compounds linked by a threemethylene link (III) were synthetised and studied as reversible inhibitors of human erythrocyte acetylcholinesterase (AChE) and as protectors against phosphorylation of the enzyme by the warfare agents Soman and VX. All the three compounds were found to be reversible inhibitors of AChE. The substrate was acetylthiocholine. The enzyme/inhibitor dissociation constants for the catalytic site were evaluated at low substrate concentrations up to 1.0 mM ; the compound II showed the highest affinity. At substrate concentrations above 1 mM inhibition by compound I revealed a second binding site. Inhibition at high substrate concentrations by the compounds II and III could not be measured due to a non-enzymic hydrolysis of the substrate by the oxime. All three compounds protected AChE against phosphorylation by soman and VX. Protection by compound I was enhanced through interaction of this compound with the allosteric binding site.

In this paper, new synthesized oxime derivatives of imidazoles and quinuclidines were tested in vitro using human erythrocyte AChE inhibited by soman and in vivo using soman poisoned mice. The inhibitory power (IC50, concentration of the tes compounds, oximes, that inhibit to 50 % of the AChE activity), acute toxicity (LD50), as well as reactivating and protective capacities, with respect to soman-inhibited AChE were tested for each of the synthesized oximes. Antidotal characteristics of the new synthesized oximes were compared and related to their chemical structures. Derivatives of imidazoles demonstrated mostly weak reactivatin and protective characteristics, both for the in vitro and in vivo cases. These characteristics were indempendent of the substituents on N phenyl imidazolium oximes. Of the imidazoles, only BMR-3 oxime strongly reactivated the soman-inhibited AChE (55%), i.e., in vitro, for human erythrocutes. BMR-4 oxime, in combination with atropine sulfate, provided effective protection in vivo (for mice)against 1.8 and 2.2 LD 50 of soman. Ont he contrary, all tested quinuclidine oximes showed very good protection in vivo (for example, BM-1 protects agains 4 LD 50 of soman), but were ineffective in vitro, i.e., against soman-inhibited human erythrocyte AChE. The results indicate that in vivo effectiveness of quinuclidine oximes against soman poisoning is not related to their reactivating or protective potentials for AChE measured in in vitro experiments ; their good protective effect is more likely to be related to other mechanisms of the cholinergic system.

Ovaj diplomski rad sastoji se od istraživačkoga i metodičkog dijela. U istraživačkom dijelu opisana je priprava O-metiloksima kinuklidin-3-ona klasičnom sintezom te primjenom mehanokemije i mikrovalnog zračenja. Spoj je pripravljen reakcijom hidrokloridnih soli kinuklidin-3-ona i metoksiamina bez dodatka baze. Kvaternizacijom kinuklidinskog dušikova atoma pripravljenog Ometiloksima s metil-jodidom, benzil-bromidom te različito supstituiranim benzil-bromidima (nitro skupine i atomi halogena u meta i para položaju) pripravljena je serija od 10 novih kvaternih derivata kinuklidin-3-on-O-metiloksima. Reakcije su praćene spektroskopijom ATR. Pripravljenim spojevima koji do sada nisu opisani u literaturi, određeno je talište te su strukture dobivenih spojeva potvrđene spektrometrijom masa, infracrvenom spektroskopijom, te 1D i 2D spektroskopijom NMR. Cilj metodičkog dijela rada je potaknuti nastavnike kemije na kreativnost u nastavi temeljenoj na učenju otkrivanjem unutar koncepta Enzimi. Navedena tema je izborni sadržaj u novom kurikulumu Kemije četvrtoga razreda gimnazije. Osmišljen je pokus na temelju kojega su oblikovani metodičkodidaktički materijali za jedan 90-minutni nastavni sat. Pri tome su postavljeni odgojno-obrazovni ciljevi temeljeni na prethodnim učeničkim znanjima, vještinama i sposobnostima. This thesis consists of a research and methodological part. The research part describes the preparation of O-methyloxime of quinuclidin-3-one by classical synthesis and the application of mechanochemistry and microwave radiation. The compound was prepared by the reaction of hydrochloride salts of quinuclidin-3-one and methoxyamine without the addition of a base. By quaternization of the quinuclidine nitrogen atom of prepared O-methyloxime with methyl iodide, benzyl bromide and differently substituted benzyl bromides (nitro groups and halogen atoms in the meta and para positions), a series of 10 new quaternary derivatives of quinuclidine-3-one-O-methyloxime was prepared. The reactions were monitored by ATR spectroscopy. The melting points of the prepared compounds, which have not been described in the literature so far, was determined, and the structures of the obtained compounds were confirmed by mass spectrometry, infrared spectroscopy, and 1D and 2D NMR spectroscopy. The goal of the methodological part of thesis is to encourage chemistry teachers to be creative in teaching based on learning through discovery within the concept of Enzymes. The mentioned topic is an optional content in the new chemistry curriculum of the fourth grade of high school. An experiment was designed on the basis of which methodological and didactic materials were designed for one 90-minute lesson. In doing so, educational goals were set based on previous student knowledge, skills and abilities.

The cholinesterase reactivators (so-called "oximes”) are used as causal antidotes in case of organophosphorus intoxications. The effectiveness of the reactivator is strongly dependent on the formation of active nucleophile, the oximate anion [1]. Its formation can be supported by optimizing the physical-chemical properties (i.e. pKa) of the oxime and the decreased pKa should lead to enhanced reactivation of phosphylated cholinesterases. For this reason, the charged chlorinated oximes were introduced and proved to highly effective in vitro for reactivation of acetylcholinesterase [2] and butyrylcholinesterase [3] inhibited by multiple organophosphorus agents. The chlorinated oximes were also proved to be not cytotoxic and effective after in vivo administration. More recently, the charged fluorinated oximes were prepared and thoroughly evaluated with promising reactivation results against multiple organophosphorus agents, although their particular lower stability was found [5]. Finally, further modified halogenated oximes with optimized oximate formation were designed and evaluated on cholinesterases inhibited by nerve agent surrogates. Some modified reactivators were proved to be in vitro effective for reactivation of NEMP, NIMP or NEDPA-inhibited AChE and surprisingly one oxime resulted as excellent reactivator of NEMP and NIMP-inhibited BChE. These promising results make prospects for further detailed investigation of modified oximes nucleophiles. This work was supported by Czech Science Foundation (no. 21- 03000S).

The Na+ /K+ -ATPase (NKA) is an enzyme that asymmetrically distributes Na+ and K+ ions across the plasma membrane to generate and maintain the membrane potential, also having a role in signaling processes. NKA dysfunction has been implicated in several neurodegenerative disorders. Its positioning as well as different functions of active and inactive pools depend on interactions with neighboring membrane lipids. Ganglioside enriched lipid rafts (LR) are housing active NKA pool, while the bulk membrane contains the inactive pool. Gangliosides are known to modulate the structure, function and localization of membrane proteins thus having an impact on ion homeostasis. The aim of this study was to investigate the effect of altered ganglioside composition on activity, protein expression and submembrane localization of NKA, using St8sia1 null mice with impaired synthesis of gangliosides. Adult wild type (WT) and null mice littermates were sacrificed, brains neuroanatomically dissected and cortical and cerebellar homogenates prepared. NKA activity was measured spectrophotometrically. Protein expression of NKA in homogenates was analyzed by Western blotting. LR and non-raft (nLR) fractions from cortices and cerebella were isolated by ultracentrifugation in discontinuous sucrose gradients, and submembrane localization of NKA analyzed by Western blotting. Data revealed statistically lower NKA activity in cortices of null mice compared to the WT mice, whilst there was no disparity in the cerebella. Total protein amount of NKA was statistically lower in null mice cortices compared to their WTs, whilst it was unvaried in the cerebella. Analysis of submembrane localization has shown higher amount of NKA to be positioned within LR of cortices than those derived from the cerebella. These results demonstrate that altered ganglioside composition may contribute to lower NKA expression influencing NKA activity and cellular ion homeostasis.

U okviru ovog rada pripravljena je knjižnica od 63 derivata potencijalno bioaktivnih oksima, etera i karbamata oksima, te α-acilamino-amida koji sadrže imidazolijevu ili kinuklidinijevu jezgru. Dodatno, sintetizirani su i α-acilamino-amidi koji sadrže i druge heterocikličke podjedinice (piridin, indol, tetrazol i tiofen) kako bi se dobila što raznovrsnija knjižnica novih spojeva koji do sada nisu opisani u literaturi. U sintezi spojeva primijenjene su klasične metode sinteze, mehanokemijske i sinteze potpomognutim mikrovalnim zračenjem. Varirani su uvjeti te uspoređene brzine i iskorištenja reakcija, ali i stereokemija nastalih produkata kod etera oksima. Optimirani su uvjeti za stereospecifičnu sintezu četiri etera oksima kinuklidina mehanokemijskom sintezom bez otapala i uz natrijev hidroksid kao bazu (nastaje anti izomer). Za određivanje omjera izomera etera oksima te konformera α-acilamino-amida u otopini primijenjena je spektroskopija nuklearne magnetske rezonancije. Mehanizam i stereokemija nastanka etera oksima kinuklidin-3-ona te konformacijski prostor α-acilamino-amida studirani su kvantno-kemijskim proračunima. In this work, a library of 63 derivatives of potentially bioactive oximes, oxime ethers, oximes carbamates, and α-acylamino-amides containing an imidazolium and/or quinuclidinium nucleus was prepared. In addition, α-acylamino-amides containing other heterocyclic subunits (pyridine, indole tetrazole and thiophene) were also synthesized in order to obtain the most diverse library of new compounds that have not been described in the literature so far. Classical methods of synthesis in solution, mechanochemical and microwave synthesis were used for the synthesis of compounds. The conditions were varied and the speed and yields of the reactions were compared, as well as the stereochemistry of the products formed in the case of ether oximes. The conditions for the stereospecific synthesis of four quinuclidine oxime ethers were optimized by mechanochemical synthesis without solvent and with sodium hydroxide as a base (anti isomer is formed). Nuclear magnetic resonance spectroscopy was used to determine the ratio of ether oxime isomers and α-acylamino-amide conformers in the solution. The mechanism and stereochemistry of quinuclidin-3-one oxime ether formation and the conformational space of α-acylamino-amide were studied by quantum-chemical calculations.