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U ovom su radu istražene mogućnosti primjene metode LC-SPE/krio NMR za praćenje kemijskih reakcija i određivanje strukture sporednih produkata i/ili razgradnih produkata on-line koji nastaju u reakciji, s naglaskom na bioaktivne molekule i lijekove. Za razvoj metodologije odabrana su tri modelna sustava. Za svaki modelni sustav istraženi su uvjeti kromatografskog odjeljivanja komponenata reakcijskih smjesâ, zatim parametri za učinkovitu ekstrakciju odabranih analita na SPE-sorbense te utjecaj parametara ekstrakcije na osjetljivost krio-probe NMR. Za identifikaciju i strukturnu karakterizaciju izoliranih spojeva korištene su spektroskopija NMR i tehnika LC-MS. Prvi modelni sustav bili su karbohidrazidi i njihovi derivati koji imaju kelirajuća svojstva te antioksidativno i antiepileptičko djelovanje. Tijekom analize karbohidrazida istražena je osjetljivost sustava LC-SPE/krio NMR. Drugi modelni sustav bio je etodolak koji pripada skupini nesteroidnih protuupalnih lijekova. Analizom 7-etiltriptofola, polazne tvari u sintezi etodolaka, identificirano je i strukturno okarakterizirano 17 onečišćenja. Treći modelni sustav bili su makrozoni, novi amidni konjugati azitromicina i derivata tiosemikarbazida, koji imaju zadovoljavajuće antibakterijsko djelovanje na odabrane susceptibilne i rezistentne bakterijske sojeve. Analizirano je ukupno 6 reakcijskih smjesâ iz triju različitih klasa novosintetiziranih makrozona. Identificirana onečišćenja bila su sporedni produkti i razgradni produkti makrolidne i tiosemikarbazonske strukture, te dijastereomeri makrozona. Konfiguracije dijastereomerâ predložene su na temelju analize spektara NOESY i računskih simulacija molekulske dinamike. Provedeno istraživanje rezultiralo je razvojem pouzdane, brže i učinkovitije metode za analizu složenih smjesa spojeva koristeći pritom znatno manje količine uzorka i organskih otapala u usporedbi s postojećim metodama off-line. Metoda LC-SPE/krio NMR može imati širu primjenu u ekstrakciji, pročišćavanju i karakterizaciji novosintetiziranih spojeva i/ili onečišćenja iz reakcijskih smjesâ. In this doctoral thesis, the possibilities of applying the LC-SPE/cryo NMR method for monitoring chemical reactions and determining the structure of by-products and/or degradation products on-line, with an emphasis on bioactive molecules and drugs, were explored. Three different model systems were explored for methodology development. For each model system, the parameters for chromatographic separation of the reaction mixtures components, as well as parameters for the efficient extraction of the selected analytes on SPE-sorbents, were optimized. Hence, the influence of the SPE-parameters on the NMR cryoprobe sensitivity was also evaluated. NMR spectroscopy and LC-MS were used for identification and structural characterization of the extracted mixture components. The first model system were carbohydrazides and their derivatives, which are known as chelating reagents and also show antioxidative and antiepileptic effects. Sensitivity of LC-SPE/cryo NMR system was determined by analyzing carbohydrazides. Etodolac, a nonsteroidal anti-inflammatory drug, was chosen as the second model system. During the analysis of 7-ethyltryptophol, a starting material in the synthesis of etodolac, 17 impurities were identified and structurally characterized. The third model system were macrozones, new amide conjugates of azitromycin and thiosemicarbazide derivatives which exhibit good antibacterial activity against susceptible and resistant bacterial strains. Six reaction mixtures from three different classes of newly synthesized macrozones were analysed. Identified impurities were classified as macrolide and non-macrolide related by-products and degradation products, as well as macrozone diastereomers. Configurations of diastereomers were proposed on the basis of NOESY NMR spectroscopy data and molecular dynamic simulations. This research resulted in development of a reliable, faster and more efficient methodology for complex mixture analysis using significantly smaller amount of sample and organic solvents in comparison with standard off-line methods. LC-SPE/cryo NMR method can be applied for the extraction, purification and characterization of newly synthesized compounds and/or impurities from reaction mixtures.

Macrolides belong to well-known class of antimicrobial agents widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a semi-synthetic 15-membered macrolide antibiotic derived from erythromycin possessing broad spectrum of antibacterial potency and favourable pharmacokinetics. However, bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major hazards to human health worldwide. Today, there is a high need for discovery of new antibiotics to combat resistance. Novel conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. Solution and solid-state structures of several macrozones have been studied and characterised by one- and two-dimensional NMR spectroscopy. Free and ribosome-bound conformations were further evaluated for selected macrozones with highest bioactivity. Saturation transfer difference (STD), WaterLogsy and transferred nuclear Overhauser effect (trNOE) NMR experiments coupled with molecular modelling studies provided valuable data about binding epitopes and bound conformations. The presented results serve as a good platform for discovery and design of new antiinfectives with improved overall biological effect.

Macrozones are novel, bioactive macrolide derivatives, conjugates of azithromycin and thiosemicarbazone. Macrolides, such as azithromycin are group of antibiotics which are active against Gram positive and some Gram negative bacteria. They exert their biological activity by binding to the 50S ribosome subunit. Due to the frequent and improper use of drugs bacterial resistance has emerged. There is a need to discover new, more effective antibiotics. In this work, three classes of macrozones were synthesized and characterized by NMR spectroscopy. In order to investigate mechanisms of macrozones activity, interactions with E. Coli ribosome were studied by trNOESY and STD NMR methods. Since 3D structure plays a significant role in bioactivity, free and bound state conformations of macrozones were also investigated. These results should contribute to discover and design new compounds with improved antibacterial activity.

Macrolides represent a group of antibiotics whose structure is characterized by a macrocyclic lactone ring linked to one or more sugars. Macrolides are bacteriostatic antibiotics, they bind to the 50S subunit of the bacterial ribosome and thus inhibit synthesis of proteins essential for the growth of bacteria. They are effective against Gram-positive and some Gram-negative bacteria. Azithromycin is a semi-synthetic derivative of erythromycin A and belongs to the group of azalides, 15-membered macrolide antibiotics. Azithromycin has excellent pharmacokinetic properties and demonstrates satisfactory biological activity. However, bacterial resistance has emerged due to the frequent use of drugs and there is a need to discover new, more effective antibiotics. As part of this study, macrozones (novel conjugates of azithromycin and thiosemicarbazone) were successfully prepared. Since thiosemicarbazones exhibit antitumor and antibacterial activity, they were used to chemically modify azithromycin at three different positions. Structure and biological activity of macrozones against susceptible and resistant bacterial strains were determined. They showed very good activities against susceptible S. pneumoniae, S. pyogenes and E. faecalis strains, and excellent activity against resistant S. pneumoniae and S. aureus strains. NMR spectroscopy was used to study interactions of prepared compounds with ribosome and to determine the binding epitopes.

Azithromycin belongs to macrolide class of antibiotics wich exert its biological activity by binding to bacterial ribosome at or near the peptidyl transferase center thus inhibiting protein biosynthesis. They are effective in treating infections caused by Gram-positive and some Gram-negative bacteria. Due to the increased development of bacterial resistance much effort is directed towards the discovery of new more potent antibiotics. A series of bioactive azithromycin derivatives, the new class of macrozones have been prepared by coupling thiosemicarbazones to azithromycin at position 4" (Figure 1). Here we present their synthesis, characterization and interactions of macrozone precursor, 4"-aminopropyl azithromycin with the ribosome isolated from E. Coli. A combination of NMR experiments such as saturation transfer difference (STD) and transferred nuclear Overhauser effect spectroscopy (trNOESY) and molecular modeling were applied to determine binding epitopes and explore bound conformations.

Makrolidi su skupina antibiotika koji se koriste za liječenje infekcija uzrokovanih Gram-pozitivnim i Gram-negativnim bakterijama.1 Pripadaju skupini bakteriostatika, vežu se za podjedinicu bakterijskog ribosoma 50S i tako inhibiraju sintezu proteina. Jedan od najpoznatijih predstavnika makrolida jest azitromicin, polusintetski derivat eritromicina A, prvog izoliranog makrolida. Azitromicin je antibiotik širokog spektra djelovanja, te je posebice djelotvoran u liječenju bolesti koje zahvaćaju respiratorni sustav. Međutim, zbog sve većeg porasta broja rezistentnih patogenih mikroorganizama potrebno je istražiti i pripraviti nove derivate makrolida koji će pokazati širu biološku aktivnost.2, 3 Makrozoni su novi konjugati azitromicina i tiosemikarbazona koji pokazuju obećavajuću in vitro antibakterijsku aktivnost. Ovaj rad obuhvaća pripravu novih makrozonskih derivata makrolidnog antibiotika azitromicina u položaju 4" (slika 1), njihovu identifikaciju i strukturnu karakterizaciju. U tu svrhu koristile su se analitičke i spektroskopske metode: tekućinska kromatografija visoke djelotvornosti, nuklearna magnetna rezonancija i spektrometrija masa.

Increasing bacterial resistance to existing antibiotics presents a serious threat to human health, and new antibacterial agents are desperately needed. Unfortunately, the number of newly marketed antibiotics has decreased dramatically in recent years. Withdrawal of the macrolide antibiotic telithromycin and inability of solithromycin to gain marketing approval have prompted our efforts to search for new anti-infective macrolide compounds. Here we present the design, synthesis and biological evaluation of a novel hybrid class of azithromycin conjugates, the macrozones. Evaluation of prepared compounds against a panel of pathogenic bacteria revealed that these molecules showed excellent activities against susceptible Streptococcus pneumoniae, Streptococcus pyogenes and Enterococcus faecalis strains comparable with or better than azithromycin. Furthermore, prepared macrozones exhibited excellent activity against efflux resistant S.pneumoniae, which was 32 times better than that of azithromycin, and very good activity against efflux resistant Staphylococcus aureus strain against which azithromycin is inactive. The results described here can serve as a good basis to guide further activities directed toward the discovery of more potent macrolide anti-infectives.

Due to a global problem of growing bacterial resistance, discovery of new antibiotics is of an outmost importance and various studies are in progress to overcome the resistance mechanisms.[1- 3] Macrozones are novel bioactive conjugates of azithromycin and thiosemicarbazides that posses very good in vitro antibacterial activity against selected Gram-positive and Gram-negative bacteria. [4] Efficient and rapid purification and isolation of newly synthesized bioactive compounds is a crucial step prior to biological evaluation. Furthermore, isolation and identification of impurities is very important procedure during the drug development in pharmaceutical industry. Preparative and semipreparative HPLC techniques are classical purification methods, but they can be time and solvent consuming. In this study we have successfully used hyphenated LC-SPE/NMR approach for the isolation and structural characterization of main components and some impurities in 9a- and 4''- tetrahydrofurfuryl macrozone reaction mixtures. Structures of the isolated compounds were proposed on the basis of the analysed one- and two-dimensional NMR spectra and MS spectra, respectively.

Macrolide antibiotics, such as azithromycin have been widely used to treat infections of both gram- positive and gram-negative bacteria. They exert their biological activity by binding to bacterial ribosome at or near the peptidyl transferase center thus inhibiting protein biosynthesis. Emerging multi-drug resistant microbial pathogens present today a serious and challenging problem in medical treatment which demands novel antimicrobial agents to be obtained. In order to discover and design more effective macrolide compounds, it is important to explore the interactions with ribosome and other biological targets and to elucidate and characterize bound conformations in solution. We have prepared a series of bioactive azithromycin conjugates, the macrozones, by coupling thiosemicarbazones to azithromycin. Here we present results of interaction studies of a selected macrozone and ribosome isolated from E. Coli. We used a combination of NMR experiments such as transferred nuclear Overhauser effect spectroscopy (trNOESY) and saturation transfer difference (STD) and molecular modeling to characterize binding epitopes and asses bound conformations.

Macrolide antibiotics, such as azithromycin, have been widely prescribed for the treatment of respiratory tract infections owing to their high efficacy and safety.[1] Azithromycin belongs to an azalide subclass of 15-membered macrolide antibiotics. Emerging multi-drug resistant microbial pathogens present today a serious and challenging global problem which demands novel and more effective antimicrobial agents to be discovered. Thiosemicarbazones and some of their metal complexes have been shown to possess anti- infective, anti-inflammatory and antitumor activities. Some thiosemicarbazones have proven very useful to treat infections, such as the new antibiotics perchlozone and thiacetazone, which are good new agents against tuberculosis.[2] Our earlier experiments which included synthesis and biological and structural characterization of some macrolide conjugates showed that those hybrid compounds exhibited good activity against some resistant bacterial strains.[3-4] Here, we present synthesis and characterization of novel macrolide conjugates, the macrozones by linking azithromycin and some selected thiosemicarbazones.

Azithromycin is the most widely used broad- spectrum antibiotic due to it's exceptional therapeutic properties.[1] Despite to this fact, an increasing number of antibiotic- resistant bacteria demands more effective antimicrobial agents to be synthesized.[2] Third generation of macrolide antibiotics still lacks activity against some Gram-negative bacterial strains, especially against respiratory tract pathogens. In order to improve antibacterial activity, new synthetic approaches were described in the literature.[3] Conjugation of the azithromycin analogue, 9a- (γ-aminopropyl) derivative, and thiosemicarbazide moieties resulted in novel compounds, macrozones, which showed good in vitro activity against selected Gram positive and Gram negative bacterial strains. Preparative and semi-preparative liquid chromatography are classical methods for purification and isolation of the final product, but they are time and solvent consuming. Modern hyphenated NMR techniques, such as LC-SPE/cryoNMR, are much faster, more efficient and more sensitive tool for the isolation and identification of the reaction mixture components. [4, 5] In this study LC- SPE/cryoNMR technique was used for the isolation of a novel ethyl-phenyl macrozone and for impurity profiling of the reaction mixture.

As impurities define the quality of a chemical entity, their identification is an essential part in the drug development process. 3-Bromo-5- (trifluoromethyl)aniline is used as a starting material in the synthesis of active pharmaceutical ingredients (APIs). In the present work, we developed and applied liquid chromatography (LC) – solid-phase extraction (SPE) / nuclear magnetic resonance (NMR) methodology with cryoprobe for separation, isolation, trapping and structural characterization of process impurities of 3-bromo- 5-(trifluoromethyl)aniline. More than fifteen analytes were separated and isolated by using LC- SPE, and structural characterization was performed on ten isolated impurities. The structures of isolated impurities were elucidated by interpretation of their one-dimensional (1H and 19F) and two-dimensional homo- and heteronuclear (COSY, 1H-13C HSQC and 1H-13C HMBC) NMR spectra and mass spectrometry (MS) data. The results demonstrated that LC-SPE/NMR system with cryoprobe can be used successfully for rapid isolation and identification of impurities of 3-bromo-5- (trifluoromethyl)aniline.

Azithromycin belongs to an azalide subclass of 15- membered macrolide antibiotics. With a much improved pharmacokinetic and therapeutic properties over erythromycin, azithromycin became the most widely used broad-spectrum antibiotic. [1] An increasing number of antibiotic-resistant bacteria, especially respiratory tract pathogens, demand a more effective antimicrobial agents to be synthesized.[2] Thiosemicarbazones belong to a large group of thiourea derivatives and are well known as antibacterial, antiviral, anti- inflammatory, antifungal and anticancer therapeutics.[3] Conjugation of the azithromycin analogue, 9a- (γ-aminopropyl) derivative, and thiosemicarbazone moieties resulted in novel compounds, macrozones, which showed good in vitro activity against selected Gram positive and Gram negative bacterial strains. Modern approach to the impurity profiling of bioactive compounds is based on the use of hyphenated systems, such as LC-NMR and/or LC- MS.[4-6] One of the most efficient and powerful tools for on-line isolation and identification of compounds in complex mixtures in the pharmaceutical industry is the LC-SPE-NMR system.[6-7] In this study, LC-SPE/NMR technique was used for impurity profiling of the novel macrozones.

Tehnika LC-SPE-NMR predstavlja suvremen spregnuti analitički sustav koji u posljednih dvadeset godina sve više pronalazi svoju primjenu u analizi farmaceutika, metabolita, prirodnih spojeva, razgradnih produkata te onečišćenja.[1] Sprega tekućinske kromatografije (engl. Liquid Chromatography, LC) s ekstrakcijom na čvrstoj fazi (engl. Solid-Phase Extraction, SPE) omogućuje učinkovito odjeljivanje komponenata smjese na kromatografskoj koloni, zatim izdvajanje i ukoncentriravanje svakog pojedinog analita na SPE- nosaču (engl. SPE-cartridge), nakon čega slijedi identifikacija i određivanje strukture izoliranog spoja spektroskopijom NMR. Prednosti ove tehnike su brojne. Zbog kvantitativnog obogaćivanja analita na SPE-nosaču, koje se postiže jednostrukim (engl. single-trapping) ili višestrukim (engl. multi-trapping) izdvajanjem analita na pojedini SPE-nosač, veća je osjetljivost mjerenja krio NMR probom.[2] S obzirom da se u kromatografskom odjeljivanju mogu koristiti nedeuterirana otapala, analiza je jeftinija, osjetljivija i učinkovitija u odnosu na tehniku LC-NMR. Sušenjem SPE-nosača u struji dušika uklanja se većina otapala iz kromatografske analize pa su signali otapala u spektru NMR reducirani. Cilj je ovog istraživanja razviti on- line LC-SPE krio NMR metodologiju koja će omogućiti praćenje kemijskih reakcija[3] te određivanje strukture nusprodukata i/ili onečišćenja koja nastaju u sintezi aktivnih farmaceutskih pripravaka[4] (engl. Active Pharmaceutical Ingredient, API) i novih derivata makrolida[5]. U ovom izlaganju bit će prikazana uspješna primjena metodologije LC-SPE-NMR u analizi onečišćenja 7- etiltriptofola, polaznog spoja u sintezi nesteroidnog protuupalnog lijeka etodolaka, te za praćenje sinteze makrozona - novih bioaktivnih konjugata azitromicina.

Macrolide antibiotics are effective and well- tolerated therapeutic agents for treating infectious diseases owing to their high efficacy and safety [1]. They have been in widespread clinical use for over 60 years and are effective against Gram-positive and certain Gram-negative microorganisms. Macrolides exert their biological activity by binding to the 50S bacterial ribosome subunit at early stage of the translation process. They bind to the ribosomal 23S rRNA in domain V at or near the peptidyl transferase region and block the exit tunnel through which the nascent peptides leave the ribosome. However, in spite of a number of existing antibiotics, the emerging multi-drug resistant microbial pathogens present serious and challenging problems in medical treatment which demand novel and more effective antimicrobial agents to be discovered. We have shown that NMR spectroscopy could reveal valuable data on free and bound state macrolide conformations [2-4] which can guide efforts directed to discovery of novel, more potent inhibitors. Here we present our recent NMR studies of new macrolide conjugates, the macrozones prepared by coupling of azithromycin and thiosemicarbazones. Macrozones have shown good activity against Gram positive strains, some Gram negative strains and also against efflux-mediated resistant Streptococcus pneumoniae. In order to characterize binding epitopes and asses bound conformations of macrozones we applied a combination of NMR experiments such as transferred nuclear Overhauser effect spectroscopy (trNOESY) and saturation transfer difference (STD) and molecular modeling

Azithromycin belongs to an azalide subclass of 15- membered macrolide antibiotics. It was synthesized in the early 1980s as a semi- synthetic derivative of erythromycin.[1] With a much improved pharmacokinetic properties over erythromycin, azithromycin became the most widely used broad- spectrum antibiotic.[2] Thiosemicarbazones belong to a large group of thiourea derivatives and are well known as antibacterial, antiviral, anti- inflammatory, antifungal and anticancer therapeutics.[3] Furthermore, a new multi- functional thiosemicarbazones were designed to treat Alzheimer and malaria.[4, 5] Conjugation of the azithromycin and thiosemicarbazone derivatives resulted in novel compounds which showed good activity against some Gram positive and Gram negative bacterial strains. Modern approach to the drug impurity profiling is based on the use of hyphenated systems, such as LC-NMR and/or LC-MS. [6] One of the most efficient and powerful tools for on-line isolation and identification of compounds in complex mixtures in the pharmaceutical industry is the LC-SPE- NMR system. In this study, LC- SPE/CRYO NMR technique was used for impurity profiling of novel azithromycin conjugates.

Impurities define the quality of a chemical entity so their unambiguous structure determination is essential part of the pharmaceutical development. Liquid chromatography (LC) – solid-phase extraction (SPE) / nuclear magnetic resonance (NMR) methodology with cryoprobe was developed and applied for identification and structural characterization of unknown impurities in 3-bromo- 5-(trifluoromethyl)aniline. The three main impurities were separated and isolated by LC-SPE system. After multiple trapping, isolated impurities were eluted from the SPE cartridges with deuterated acetonitrile and one- and two- dimensional homo- and heteronuclear NMR spectra were recorded. The structures of the unknown impurities were determined by detailed inspection of NMR spectra and by mass spectrometric (MS) analysis to show that the impurities in question are di-bromo derivatives of 3- (trifluoromethyl)aniline. The results demonstrated that LC-SPE / NMR can be used for rapid impurity profiling of 3-bromo-5-(trifluoromethyl)aniline.

7-Ethyltryptophol, 2(7-ethyl-1H-indol-3- yl)ethanol, is a key starting material in the synthesis of Etodolac, a non-steroidal anti- inflammatory drug. Depending on the synthetic pathway for 7-ethyltryptophol, commercially available material comprises many different impurities which can cause formation of coproducts in the synthesis of Etodolac thus complicating the purification of the final product.[1] Therefore, to develop an optimal purification procedure of Etodolac, it is important to know the structures of impurities in 7-ethyltryptophol. Classical methods for separation and isolation of impurities, such as preparative or semi- preparative liquid chromatography, are time and solvent consuming. Nowadays, hyphenated NMR techniques are becoming faster, more efficient and more sensitive tool for determination of impurities and degradation products in pharmaceuticals and natural products. [2, 3] In this study LC-SPE (cryo)NMR methodology was used to identify impurities in 7-ethyltryptophol. Chromatographic separation was achieved on Waters XBridge Phenyl and C18 columns (150 mm x 4.6 mm ; 3.5 μm) using a combination of gradient and isocratic elution. Compounds were trapped on HySphere Resin GP cartridges in a SPE multitrapping mode. 1H, COSY, 1H-13C HSQC, 1H-13C HMBC NMR spectra and MS spectra were recorded to determine the structures of impurities. In this way 17 compounds were identified. These results show a good potential of LC-SPE (cryo)NMR technique in an identification and structural characterization of low level impurities and degradation products in pharmaceuticals. References: [1] M. C. Sekharayya, G. V. Narayana, S. Nigam, G. Madhusudhan, Indian J. Chem. 51B (2012) 1763-1766. [2] S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R. P. Shah, J. Pharm. Biomed. Anal. 69 (2012) 148-173. [3] J. W. Jaroszewski, Planta Med. 71 (2005) 795-802

7-Ethyltryptophol, 2(7-ethyl-1H-indol-3- yl)ethanol, is a key starting material in the synthesis of Etodolac, a non-steroidal anti- inflammatory drug (Scheme 1). Depending on the synthetic pathway for 7- ethyltryptophol, commercially available material comprises many different impurities which can cause formation of coproducts in the synthesis of Etodolac thus complicating the purification of the final product.[1] Therefore, to develop an optimal purification procedure of Etodolac, it is important to know the structures of impurities in 7- ethyltryptophol. LC-SPE-NMR methodology is a powerful tool for fast identification and structural elucidation of low concentration level impurities and degradation products in pharmaceuticals.[2] In this study we report on the HPLC method for efficient separation of impurities in 7- ethyltryptophol as the first step in hyphenated LC-SPE-NMR analysis. References 1. M. C. Sekharayya, G. V. Narayana, S. Nigam, G. Madhusudhan, Indian J. Chem. 51B (2012) 1763-1766. 2. S. Singh, T. Handa, M. Narayanam, A. Sahu, M. Junwal, R. P. Shah, J. Pharm. Biomed. Anal. 69 (2012) 148-173. Acknowledgements: This work was funded by European Regional Development Fund and Croatian state budget (project Met4Pharm).

A microwave-assisted extraction procedure was designed for the extraction of phenylurea herbicides isoproturon, chlortoluron, diuron and linuron, triazine herbicide terbuthylazine, and its degradation product deethylterbuthylazine from soil. The sample preparation was optimized for final analysis by high-performance liquid chromatography with a UV diode array detector. The limit of detection for all the analytes in standard mixture prepared in deionized water was 5 ng/ml, whereas the limit of quantification was 18 ng/ml. The effects of the extraction solvent, temperature, and water content in soil on microwave extraction efficiency of triazines and phenylurea herbicides from soil were investigated by analysing spiked soil samples. The analytes were added to a forest soil in mass fractions ranging from 93 to 113 ng/g. The best results were achieved by extraction of soil with solvent mixture ψ(acetone, n-hexane)= 1:1 at 60 °C and at soil water content of 20 %. The extraction efficiency from Cmrok wet soil was 84 % for isoproturon, 80 % for chlortoluron, 60 % for diuron, 85 % for linuron, 93 % for terbuthylazine, and 95 % for dethylterbuthylazine. Extraction efficiency and reproducibility of triazine and phenylurea herbicides with ψ(acetone, n-hexane) = 1:1 at 60 °C was also tested by extracting analytes from two agricultural dry and wet soils to which they were added at different mass fraction levels ranging from 37 to 45 ng/g, from 93 to 113 ng/g, and from 374 to 450 ng/g. The extraction efficiency of analytes was not dependent on their mass fractions in soil. The recoveries ranged from 72 % for isoproturon and diuron to 79 % for deethylterbuthylazine with relative standard deviation between 7 and 10 %. The limit of detection for all of the analytes in soil was 10 ng/g.

Aromatic Schiff bases derived from carbohydrazide possess a broad spectrum of pharmacological activities and a wide variety of applications in many fields, such as inorganic, analytical and organic chemistry. They have been used as multitopic ligands for the targeted construction of bioactive coordination systems, as selective analytical reagents for ion binding and as precursors in the syntheses of nitrogen containing heterocycles. Schiff bases can easily be prepared by the condensation reaction between appropriate primary amine and carbonyl compounds. The condensation of carbohydrazide with salicylaldehyde in different solvents yielded a mixture of mono- (1) and bis(salicylidene)carbohydrazide (2). Structure characterization of the reaction products in solution and solid state was performed by off-line NMR, Raman and SWAXS analysis. It has been demonstrated that the ratio of 1 and 2 and the polymorphic content of 2 are dependent on solvent used for the reaction. In order to precisely estimate the product ratio and reaction end point in real time, the process was also monitored on- line by a coupled LC-NMR system.

Etodolac, 1, 8-diethyl-1, 3, 4, 9-tetrahydropyrano- [3, 4-b]indole-1-acetic acid is a non-steroidal antiinflamatory antirheumatic drug. A key intermediate in the synthesis of Etodolac is its methyl ester which can be obtained by oxa- Pictet-Spengler reaction, starting from 7- ethyltriptophol. Pyrano[3, 4-b]indole ring is closed in the reaction of 7-ethyltriptophol and β-ketoester, methyl 3-oxo-pentanoate. For the preparation of oxacycle (pyranic ring) different Brönsted and Lewis acids can be employed.[1] Typically, aqueous hydrochloric or sulfuric acid, as well as boron trifuoride etherate, gasous hydrochloride, p- toluensulfonic acid, zink chloride, aluminium chloride and tin(IV) chloride in organic solvents are used to promote the reaction. Catalyst such as boron trifuoride etherate is expensive and is stable only in a perfectly anhydrous environment, which is difficult to manage, especially in productions on a larger scale. Therefore, the processes in which inorganic mineral acids can be used are preferred.[2] We report the kinetic study of conversion of 7-ethyltriptophol to methyl ester of Etodolac in methanol in the presence of concentrated hydrochloric and sulfuric acids. In order to optimize the synthetic procedure, kinetic profiles of reactions promoted with different molar ratios of acids (with respect to the β-ketoester) were determined using HPLC as a method of choice.

Etodolac methyl ester is a key intermediate in the synthesis of Etodolac, a non-steroidal anti-inflammatory drug. It can be obtained by oxa-Pictet-Spengler reaction, starting from 7- ethyltriptophol and using inorganic mineral acids as catalysts.[1] In order to get kinetic profiles of reactions catalyzed with different molar ratios of sulfuric and hydrochloric acid, a high performance liquid chromatography method with UV detection was developed for monitoring conversion of 7-ethyltryptophol to methyl ester of Etodolac. For that purpose different stationary phases (C18, Phenyl and NH2 column) were investigated for separation of 7-ethyltryptophol and Etodolac methyl ester. Elution was performed with three different solvent mixtures in isocratic and/or gradient mode. The best chromatographic separation was achieved isocratically using Waters Symmetry Shield RP 18 column with 55% acetonitrile and 45% water.

Tryptophols are derivatives of 2-(1H-indol-3-yl)ethanol, indole class bearing a C-3 hydroxyethyl side chain. 7-Ethyltryptophol, 2-(7-ethyl-1H-indol-3-yl)ethanol, is a key starting material in the synthesis of Etodolac, an important non-steroidal anti-inflammatory drug. The currently available synthetic procedures for 7-ethyltryptophol usually result with formation of a high-level of various impurities. LC-NMR is a very powerful tool for obtaining detailed structural information of components in complex mixtures such as impurities and metabolites in pharmaceuticals, natural products and synthetic polymers. In the present study, we developed LC-NMR methodology for separation and structural analysis of impurities in commercially available 7-ethyltryptophol. Several impurity peaks were detected in chromatograms and the structures of major impurities were elucidated by analyzing 1D and 2D LC-NMR spectra.

Etodolac, 1, 8-diethyl-1, 3, 4, 9- tetrahydropyrano- [3, 4-b]indole-1-acetic acid is a racemic non- steroidal anti-inflammatory antirheumatic drug. The key intermediate in Etodolac synthesis is its methyl ester which is usually obtained by oxa-Pictet-Spengler reaction, starting from commercially available chemicals 7- ethyltryptophol and methyl 3-oxo- pentanoate, using different Brönsted and Lewis acids as promoters.1 Etodolac is than obtained by subsequent saponification step usually in a polar solvent.1, 2 We have performed extensive kinetic study of oxa-Pictet-Spengler reaction with different molar equivalents of hydrochloride and sulfuric acid as promoters. The study showed that two molar equivalents of sulfuric acid are optimal for reaction progress. Based on these results, we developed the methodology in which Etodolac can be obtained in a single step. For that purpose the novel synthetic procedure for methyl ester intermediate was firstly created using in start optimal solvent (isopropyl alcohol) for subsequent hydrolysis step. The intermediate and the final product were characterized by NMR spectroscopy. The combination of LC and NMR in an on-line and off-line mode was applied for structural elucidation of reaction impurities which may originate from starting material or are formed in methyl ester intermediate synthesis. The approach described in this study contributed to the development of more efficient synthetic procedure for the preparation of Etodolac. References 1. E. L. Larghi, T. S. Kaufman, Synthesis (2006) 187– 220. 2. E. Vigano, P.Colombo, U.S. patent (2000) US 6066741 A 20000523. Acknowledgements: This work was funded by European Regional Development Fund and Croatian state budget (project Met4Pharm).

Razrađeni su uvjeti mikrovalne ekstrakcije fenilurea-herbicida izoproturona, klortolurona, diurona i linurona, triazinskog herbicida terbutilazina i njegovog razgradnog produkta deetilterbutilazina iz tla. Priprava uzoraka optimirana je za konačnu analizu tekućinskom kromatografijom visoke djelotvornosti uz detekciju UV-detektorom s nizom dioda. Granica detekcije svih analita u standardnoj smjesi pripravljenoj u deioniziranoj vodi bila je 5 ng/ml, a granica kvantifikacije 18 ng/ml. Usporedbom nagiba kalibracijskih pravaca izračunanih regresijskom analizom kromatografskih podataka za standardne otopine pripravljene u vodi i u ekstraktima tala pokazan je utjecaj matrice uzorka na selektivnost i osjetljivost određivanja terbutilazina, deetilterbutilazina, diurona i linurona. Ispitan je utjecaj otapala/smjese otapala, temperature i vlage na djelotvornost mikrovalne ekstrakcije triazinskih i fenilurea-spojeva dodanih u tlo Cmrok u masenim udjelima od 93 ng/g do 113 ng/g. Najbolji rezultati za sve spojeve postignuti su ekstrakcijom sa smjesom otapala ψ(aceton, n- heksan) = 1:1 pri 60 °C i uz sadržaj vlage u tlu od 20 %. Djelotvornosti ekstrakcije istraživanih spojeva iz vlažnog tla Cmrok bile su 84 % za izoproturon, 80 % za klortoluron, 60 % za diuron, 85 % za linuron te 93 % za terbutilazin i 95 % za deetilterbutilazin. Djelotvornost i ponovljivost mikrovalne ekstrakcije triazinskih i fenilurea- spojeva iz tla sa smjesom otapala ψ(aceton, n- heksan) = 1:1 pri 60 °C provjerena je ekstrakcijom suhog i vlažnog tla Sapci kojem su spojevi dodani u masenim udjelima od 37 ng/g do 45 ng/g, od 93 ng/g do 113 ng/g i od 374 ng/g do 450 ng/g. Djelotvornost ekstrakcije svih spojeva iz suhog i vlažnog tla Sapci nije ovisila o njihovom masenom udjelu u tlu. Analitički povrati bili su u rasponu od 72 % za izoproturon i diuron do 79 % za deetilterbutilazin uz relativnu standardnu devijaciju od 7 % do 10 %. Granica detekcije svih određivanih spojeva u tlu Sapci bila je 10 ng/g.