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Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl- D-isoglutamine) is the smallest peptidoglycan fragment capable of replacing the whole Mycobacteria in Freund’s adjuvant. MDP acts as a pathogen-associated molecular pattern and activates the NOD2 receptor. Numerous structural variations of MDP have been performed in order to improve its pharmacological properties. The main parameter for the improvement is lipophilicity. We have designed and prepared desmuramyl peptides containing lypophilic 2-aminoadamantane-2- carboxylic acid, and furthermore modified it with mannose moieties. Mannose receptors presented on immunocompetent cells are considered to be pattern-recognition receptors, as well as NOD2, and therefore they can affect the immune reactions. Here we present the synthesis and biological evaluation of desmuramyl peptides containing 2- aminoadamantane-2-carboxylic acid, and its mono- and di-mannosylated derivatives. Their immunostimulating activities were evaluated in vivo in the mouse model using ovalbumin as an antigen. The effects on the secondary humoral response were determined by measuring overall anti-OVA IgG antibodies, and the subclasses anti- OVA IgG1 and anti-OVA IgG2a. Mannosylated desmuramyl peptides exhibited an improved immunological activity and the di-mannoslyated derivative showed to be the most active one.

Peptidoglycan fragments of well defined structures, the best known of which are muramyl peptides, have been extensively studied as possible adjuvants for human and animal vaccines. Muramyl dipeptide (MDP, N- acetylmuramyl-L-alanyl-D-isoglutamine) is known as the smallest synthetic adjuvant molecule capable of replacing whole Mycobacteria in Freund’s adjuvant.1 Mannose receptors (MR), present on immunocompetent cells (such as macrophages and dendritic cells) are considered to be pattern- recognition receptors biding compounds with mannose, N-acetylglucosamine or fucose as their essential parts. Therefore, they are responsible for the binding, among others, of mannosylated antigens or relevant biologically active molecules containing mannose, thus affecting the immune reactions.2 Up to now our research in the field of potential adjuvants was directed towards desmuramyl peptides which contain adamantylglycine and mannosylated adamantylglycine moieties bound to the essential part of MDP, L-Ala-D-isoGln.3 Here we present synthesis and biological evaluation of novel mannosylated desmuramyl peptides containing glycolyl linker. Investigation of their immunostimulating activity was conducted in vivo in mouse model using ovalbumin as an antigen. Synthesized glycopeptides showed improved immunological activity than previously described mannosylated desmuramyl peptides with (R)- hydroxyisobutyl linker.

Muramyl dipeptide (MDP), N-acetylmuramyl-L-alanyl- D-isoglutamine, is the smallest structural unit of bacterial peptidoglycan responsible for eliciting of immune response. It acts as a pathogen- associated molecular pattern and activates nucleotide-binding oligomerization domain- containing protein 2 (NOD2). [1] Desmuramyl peptides possess L-Ala-D-isoGln pharmacophore which is essential for the immunostimulatory properties. We have designed mannosylated desmuramly peptides (Figure 1) using multiple pathogen recognition receptor activation approach. [2] Mannose receptors are C-type lectin receptors expressed on immunocompetent cells and represent additional pattern recognition target for affecting immune system. [3]Adamantane was introduced in order to increase the lipophilicity of L-Ala-D-isoGln peptide and to facilitate the anchoring of the compound to the lipid bilayer.

Kojic acid is a chelating compound and a common fungal metabolite widely used primarily in the preparation of cosmetic biomaterials and skin care products [1]. Numerous studies also showed that kojic acid and its derivatives exhibit other various biological activities [2, 3]. In view of our previous findings on the adamantyl pyridinone derivatives and our continuous investigation of their biological activity [4] we wanted to extend our research further and explore the influence of the adamantyl group on the biological activity of pyranone system as well. Thus, esters of kojic and adamantan‐1‐ylacetic acid were prepared using efficient synthetic methods in good yields and evaluated for their in vitro antiproliferative activity on 4 cancer cell lines (K562, HeLa, Caco‐ 2, NCI‐H358) as well as on normal cells (MDCK). Compounds showed good to moderate in vitro antiproliferative acitvity with IC50 values ranging from 13.1 to 43.0 μM. It seems that the presence and adequate position of the adamantyl acyl group or chlorine atom is a prerequisite for their antitumor activity. The presence and the position of the adamantyl acyl unit were also the important structural parameters that governed antiproliferative activity of our previously investigated pyridin‐4‐ one derivatives.

Ugljikohidrati su važne organske makromolekule koje se tradicionalno uglavnom spominju u kontekstu izvora energije, dok se rjeđe tumače njihove esencijalne uloge u biološkim sustavima. Oligosaharidi nose informacije koje su određene mjestom vezanja monosaharida, konfiguracijom glikozidne veze (α- ili β-) te udjelom razgranatosti, što ih čini izvrsnim prenositeljima biološke informacije. Lektini su proteini koji specifično vežu šećerne strukture. Poseban tip lektina su manozni receptori koji su prisutni na površini različitih imunokompetentnih stanica, kao što su makrofagi i dendritičke stanice te su prepoznati kao potencijalne mete za vezanje manoziliranih antigena, relevantnih biološki aktivnih molekula koje sadrže manozu.1 Također se mogu koristiti za unos lijekova koji u svojoj strukturi sadrže manozu, te utjecati na sami tijek reakcija u imunološkom sustavu. Cilj ovog rada je razvoj metode priprave manozilirane karboksilne kiseline (2), važne podstrukture u sintezi spoja koji bi bio potencijalni adjuvant. Acetiliranjem D-manoze (1) (a) anhidridom octene kiseline uz jod kao katalizator, pripravljena je 1, 2, 3, 4, 6- penta-O-acetil-α, β-D- manopiranoza kojoj je selektivno je uklonjena anomerna acetatna zaštita na tri različita načina te je dobivena 2, 3, 4, 6- tetra-O-acetil-α- D-manopiranoza. U trećem koraku (c) je pripravljen O-manozid reakcijom tetraacetilirane manoze s tert-butil- bromacetatom i kalijevim karbonatom pri čemu je preferirano nastao α-anomer O-manozida. Anomerno čistom esteru uklonjena je tert- butilna zaštita pomoću trifluoroctene kiseline u suhom diklormetanu čime je pripravljena 2- (2, 3, 4, 6-tetra-O-acetil-α-D- manopiranoziloksi)octena kiselina (2) u dobrom prinosu, a strukture produkata su detaljno okarakterizirane 1H NMR i 13C NMR spektroskopijom.

Muramyl dipeptide (MDP, MurNAc-L-Ala-D-isoGln) is an immunostimulative peptide. It is the minimal structure of bacterial cell wall peptidoglycan with immunostimulatory (adjuvant) activity.[1] However, one of the major side-effects of MDP is pyrogenicity. Therefore, numerous MDP derivatives were synthesized with the aim to avoid unwanted side-effects, and many of them have a wide variety of therapeutical potentials ; clinical uses are known as well.[2] A very important parameter in the improvement of pharmacological properties of MDP is lipophilicity, e.g. it eliminates drawbacks caused by poor macrophage penetration and rapid elimination. In our previous work, we have prepared desmuramyl peptides modified at the N- terminus with the lipophilic adamantyl group as well as their mannosylated derivatives where mannose was connected to the peptide core over a lactyl linker. These compounds showed significant immunostimulating activity in experiments in vivo. [3, 4] Mannose was introduced in order to facilitate the targeting of immune cells by surface mannose receptors. In continuation of this research, we have designed novel mannosylated desmuramyl peptides in which mannose was connected to desmuramyl peptide over O-glycosyl linker and with lipophilic adamantane introduced at the peptide N- terminus. Immunomodulating properties of synthesized peptides will be evaluated in the mice model using ovalbumin as an antigen and compared with previously prepared derivatives.

Muropeptides are fragments of unique polymers that build up the cell wall of bacteria, also called peptidoglycans. Muramyl dipeptide (MDP), N- acetylmuramyl-L-alanyl-D-isoglutamine, is the smallest structural unit of peptidoglycans showing the immunostimulating (adjuvant) activity and desmuramyl peptides are MDP analogues lacking the hydrophilic carbohydrate moiety. Numerous derivatives with different groups at C- and N- terminus of the L-Ala-D- isoGln moiety are known. Part of our earlier work was based on the synthesis of adamantyl desmuramylpeptides, where lipophilic adamanatane subunit is attached to the L-Ala-D- isoGln. Inserted adamantane group facilitates the anchoring of the peptidoglycan ʻcargoʼ to the membrane and thus exhibit enhanced immunomodulating activity which was proven in in vivo experiments. Our next object of interest refers to the influence of mannosylation on immunostimulating activity of adamantyl tripeptide. Mannose is an important sugar whose receptors, mannose receptors (MRs), present on immunocompentent cells (such as macrophages and dendritic cells) are considered to be pattern- recognition receptors binding compounds with mannose. Therefore, they are responsible for the binding mannosylated antigens or other relevant biologically active molecules containing mannose, thus affecting the immune reactions. Our current research work includes developing methodology for the stereoselective synthesis of di- and tri- antennary mannose derivatives of desmuramyl peptides where we believe that increasing of mannose subunits number will enhance strength of overall mannose – lectin interactions.

Muropeptides, also called peptidoglycans, are fragments of unique polymers that build up the cell wall of bacteria. Muramyl dipeptide (MDP), N- acetylmuramyl-L-alanyl-D-isoglutamine, is the smallest structural unit of peptidoglycans showing the immunostimulating (adjuvant) activity. Dipeptide core of MDP is the essential structure required for adjuvant activity, therefore, MDP analogues lacking the hydrophilic carbohydrate moiety, desmuramyl peptides, are explored. Numerous derivatives with different groups at C- and N-terminus of the L-Ala-D- isoGln moiety are known. We have designed and synthesized desmuramyl peptides with incorporated lipophilic adamantyl group and also their mannosylated derivatives. Conformational study of desmuramyl dipeptide, its tripeptide analogue with incorporated bulky lipophylic adamantyl substituent at the N- terminal amino acid and serine derivative of adamantyl tripeptide was also performed. Attached mannose allows the targeting of cell surface receptors recognizing glycans on immune cells while adamantane group facilitates the anchoring of the peptidoglycan ‘cargo’ to the membrane. In order to improve lectin-carbohydrate binding, we are also developing methodology for the stereoselective synthesis of dimannosylated desmuramylpeptides because we believe that increasing of mannose subunits number will enhance strength of overall mannose – lectin interactions.

Adhesion of pathogenic organisms to host tissues is the initiation of the majority of infectious diseases. It is often mediated by lectins present on the surface of infectious organisms which then combine with complementary sugars on the host surface.[1] One of the best characterized bacterial lectin involved in receptor-ligand interaction is mannose-specific type 1 fimbrial FimH adhesin. For example, in uropathogenic E. coli (UPEC) mannose-specific adhesion is mediated by this lectin.[2] A large number of α-D-mannopyranosides with aromatic aglycon moiety were found to bind with high affinity to FimH and thus prevent agglutination of red blood cells. In our previous work we have explored the inhibitory potential of α- mannosides with meta and para substituted N- aryl 3-hydroxy-2-methylpyridin-4-ones as aglycon parts of a molecule.[3, 4] The hemagglutination assays revealed greater preference of FimH towards the para substituted pyridinone mannosides. In this work we report the synthesis of novel para substituted N-aryl 3-hydroxypyridin-4-ones without methyl group in position 2 of the pyridinone ring (Figure). Their inhibitory properties towards the adhesion of E. coli to quinea pig erythrocytes will be evaluated using hemagglutination assay. These results will be compared with inhibitory potencies of analogous para derivatives of N- aryl 3-hydroxy 2-methylpyridin-4-ones. Computational analysis complemented experimental results by elucidating specific interactions within the FimH active site responsible for the binding and aided in the interpretation of the observed binding trends.

Compounds containing a heterocyclic moiety attached to a carbohydrate system are important biologicaly active compounds. Pyridinium, imidazolium and benzimidazolium derivatives, among others, have been used in natural product chemistry and organic synthesis. Furthermore, they exibit a wide variety of biological activities. It is well established that the cholinesterases (ChE), particularly butyrylcholinesterase (BChE), are associated with te patogenesis and progression of Alzheimer's and Parkinson's disease.1 Cholinesterase inhibitors (ChEI) have been widely recognized as an effective palliative treatment for Alzheimer's and Parkinson's disease, preventing the breakdown of neurotrasmitter acetylcholine (ACh) by inhibiting ChE in the brain regions relevant for the above mentioned diseases.1 Many heterocyclic glycoconjugates have received a great deal of attention in this field of research. A series of novel heterocyclic β-D-gluco-, β-D- galacto- and α-D-mannopyranoside derivatives was synthesised as potential BChE inhibitors. Synthesis of target molecules was conducted in four steps: protection of sugar hydroxyl groups by peracetylation, O-glycosylation of the anomeric C-atom with 2-bromoethanol leading to the introduction of two C-atoms spacer followed by condensation with pyridine, imidazole and benzimidazole derivatives and the final removal of acetyls as protecting groups. All compounds were prepared in good yields and were characterized by standard analytical methods (1D NMR, IR, MS) and optical rotation measurements. Inhibitory properties of all synthesized compounds towards BChE were studied by applying Ellman's method.2, 3 Kinetic parameters (Km, Vm) and the inhibition constant (Ki) were determined.

Muropeptides are fragments of peptidoglycans, unique polymers that build up the cell wall of bacteria. They are used as immunostimulating compounds (adjuvants). Muramyl dipeptide (MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine, is the smallest structural unit of peptidoglycans showing the immunostimulating activity. Numerous MDP analogues lacking the hydrophilic carbohydrate moiety (desmuramyl peptides) with different groups at C- and N-terminus of the L- Ala-D-isoGln moiety have been synthesized. [1] We have designed and synthesized desmuramyl peptides with incorporated lipophilic adamantyl group and also their mannosylated derivatives. [2, 3] Attached mannose allows the targeting of cell surface receptors recognizing glycans on immune cells while adamantane group facilitates the anchoring of the peptidoglycan ‘cargo’ to the membrane. Synthesized desmuramyl peptides have been encapsulated into liposomes, vehicles often used for the target delivery. In these liposomal formulations adamantane group penetrates into the lipid bilayer while mannose is exposed at the liposome surface and can serve in targeting mannose receptors. [4] In order to improve lectin-carbohydrate binding we are developing methodology for the stereoselective synthesis of di- and tri- antennary mannose derivatives of desmuramyl peptides because increasing of mannose subunits number will enhance strength of overall mannose – lectin interactions.

Bacterial adhesion to host cell surfaces is the prerequisite for the initiation of the infectious diseases. Urinary tract infections are the prevalent inflammatory diseases mainly caused by uropathogenic E. coli (UPEC). E. coli adhesion to the bladder epithelium is mediated through recognition of the tissue-surface mannosylated proteins with bacterial lectin FimH. The inhibition of this recognition-dependent interaction has been recognized as a promising strategy for the development of an anti-adhesion therapy. The mannosides with either aryl- or elongated alkyl-functionalized aglycon portions have been shown to be potent inhibitors of FimH- mediated adhesion. Herein, we present the synthesis of new bioorganometallics 8 and 9 containing amide groups, butyl or pentyl spacer and phenylalanine as an extra aromatic unit. The biological actvity of new compounds against bacterial lectin FimH is tested with hemagglutination inhbition assay (HAI).

Click chemistry refers to fast reactions that give very high chemical yields, use easily removable solvents and mild conditions while byproducts are easily removable as well. Staudinger ligation, copper free and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) have been increasingly used for fluorescent labeling of biomolecules as well as for the synthesis of small biologically active compounds. Peptidogycans are polymeric components of bacterial cell walls and their monomers stimulate strong immune response. It has been shown that a minimal structural unit of peptidoglycan monomer showing a significant adjuvant activity is muramyldipeptide (MurNAc-L-Ala-D-isoGln, MDP). The aim of this work was the synthesis of mannosylated derivatives of desmuramylpeptides, MDP analogues lacking the original carbohydrate moiety, for further biological evaluation. Mannose moiety serves for targeting the mannose receptors on surface of the immune cells thereby enhancing the specificity of interactions with the cells thus affecting the type of immune response. In order to improve the lipophilicity of mannosylated desmuramyldipeptides, some of which have been previously shown to improve the pharmacological properties of MDP, the peptide part was conjugated with the adamantyl moiety over a triazole ring. Adamantyl substituted triazoles were prepared via CuAAC, conjugated with desmuramyldipeptides and linked to mannose via different short linkers to improve flexibility and accessibility of mannose towards mannose receptors.

3-Hydroxypyridin-4-ones are nowadays widely investigated for their broad spectrum of biological activities (antibacterial, antidiabetes, antiprotozoal, antineurodegenerative and anticancer) [1]. Recently, we prepared and investigated several N-aryl substituted 3-hydroxy-2-methylpyridin-4- ones as well as their ester adamantyl derivatives for their in vitro antitumor properties on several cancer cell lines [2]. All tested compounds showed antiproliferative activity ranging from moderate to strong on all inspected cell lines with lipophilic adamantane containing derivatives being active at low micromolar IC50 concentrations. Further studies of those derivatives in terms of their mechanism of action is now in progress. In this work, for the purpose of the structure-activity relationship study (SAR), novel lipophilic ether (adamantyl and cyclohexyl) derivatives of N-aryl substituted 3- hydroxy-2-methylpyridin- 4-ones were prepared (Figure 1) and their in vitro antitumor properties evaluated on the related panel of cancer and leukemia cell lines. The compounds were synthesized starting from corresponding pyridinones and adamant-1-yl or cyclohexyl bromoacetates by Williamson synthesis. Antitumor properties of novel compounds will start to elucidate the key elements (ester or ether bond between lipophilic unit and pyridinone part, the nature and/or the position of lipophilic unit) needed for high antiproliferative activity of observed pyridinone derivatives. In vitro testing included cytotoxicity evaluation and additional testing of the most potent candidates to determine the mechanism of action more precisely.

Muramyldipeptide (MDP), N-acetylmuramyl-L- alanyl-D-isoglutamine, is the smallest structural unit of peptidoglycans displaying immunostimulating (adjuvant) activity. Structure-activity relationship studies of muropeptides suggest that the desmuramyl dipeptide part (L-Ala-D-isoGln) is responsible for the adjuvant activity [1]. Primary structure modifications as well as their conformational changes may be responsible for changes in biological activity of peptides. Increase of lipophilicity in the N- and C- terminus of the dipeptide, such as introduced adamantly group at the N- terminal amino acid, can significantly improve pharmacological properties of desmuramyl peptides. Synthesis as well as conformational computational and experimental NMR analysis of corresponding desmuramyl dipeptide, tripeptide and tetrapeptide derivatives in zwitterionic and non-zwitterionic form was performed. L-Alanyl-D-isoglutamine was obtained by the removal of N- and C- protecting groups from commercially available dipeptide. Tri- and tetrapeptides were obtained by consecutive attachment of corresponding amino acids (1-adamantyl-glycine and serine) to desmuramyl dipeptide, using optimized coupling methodology (EDC/HOBt coupling reagents in the mixture of solvents CH2Cl2/1, 4- dioxane 1:1). (S)-1- adamantyl-glycyl-L-alanyl-D-isoglutamine was prepared in four synthetic steps using more efficient method than previously reported [2, 3]. The most stable conformers of synthesized peptides were determined. A systematic conformational space search was performed by means of molecular and quantum mechanical study in solution, which was modelled as a polarizable continuum. Hydrogen bonds were determined by using the QTAIM population analysis method. Calculated conformers were compared with those obtained in solution by NMR study in deuterated water and DMSO.

Muropeptides are fragments of peptidoglycans, unique polymers that build up the cell wall of bacteria. They are used as immunostimulating compounds (adjuvants). Muramyl dipeptide (MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine, is the smallest structural unit of peptidoglycans showing the immunostimulating activity. Numerous MDP analogues lacking the hydrophilic carbohydrate moiety (desmuramyl peptides) with different groups at C- and N-terminus of the L-Ala-D-isoGln moiety have been synthesized. We have designed and synthesized desmuramyl peptides with incorporated lipophilic adamantyl group and also their mannosylated derivatives. Attached mannose allows the targeting of cell surface receptors recognizing glycans on immune cells while adamantane group facilitates the anchoring of the peptidoglycan ‘cargo’ to the membrane. Synthesized desmuramyl peptides have been encapsulated into liposomes, vehicles often used for the target delivery. In these liposomal formulations adamantane group penetrates into the lipid bilayer while mannose is exposed at the liposome surface and can serve in targeting mannose receptors. Since mannose derived desmuramyl peptides show great biological potential, we have developed novel and improved methodology for their synthesis. Previously described procedure included stereoselective glycosylation reaction with benzylated mannose, while new method consists of stereospecific α-O-mannosylation using tetraacetylated mannopyranose. Besides stereospecificity this strategy has several other advantages such as higher yields, shorter reaction times, easier purification of products and possibilities for the preparation of prodrug formulations, even at larger scale (gram scale). Novel methodology will be presented on a synthesis of series of desmuramyl peptides.

In the last couple of decades heterocyclic N- substituted 3-hydroxy-2-methylpyridin-4-one based compounds have emerged as an important substructure found in many natural and synthetic biologically active materials.1 High selectivity towards trivalent metal cations, especially Fe(III), is the primary basis of a number of 3- hydroxypyridin-4-ones' biological activities. These compounds have high potential in the treatment of iron overload diseases, in various blood pathologies such as thalassemias and hemochromatosis as well as in the treatment of neurodegenerative diseases. Various positions on the pyridinone ring, especially nitrogen atom, can be easily modified which allows the rational design of these compounds and modulation of their specific physicochemical and biological properties. Adamantyl based compounds are used clinically primarily for the treatment of neurological conditions, as well as antiviral and antitumour agents. The bulky, hydrophobic adamantane group was shown to modulate the therapeutic activity of many experimental drugs and prodrugs by increasing their partition coefficients and also by varying their mechanism of action.2 In view of our ongoing interest in pyridinone group of compounds3, especially in their biological activity4, we report the synthesis and antiproliferative in vitro study of several N-aryl 3-hydroxy-2-methylpyridin-4-ones and their lipophilic adamantyl analogues. Cytotoxicity was tested on K562 and MCF7 cell lines and several potential candidates were identified. These compounds showed antiproliferative effect in both lines in and below micromolar concentration range. Mechanism of growth inhibition (apotosis and cell cycle distribution) was investigated for specific compounds based on their cytotoxycity and structure.

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

N-alkil i N-aril supstituirani 3- hidroksipiridin- 4-oni (3, 4-HP), skupina heterocikličkih organskih spojeva, poznati su bidentatni kelatori brojnih metala (Fe(III), Al(III), V(V) i dr.). Njihov kelirajući učinak temelj je mnogih istraživanja, bilo da je riječ o sintezi i ispitivanju terapijskog učinka njihovih derivata [1] ili spektrofotometrijskom određivanju metalnih iona [2]. U našem se laboratoriju zadnjih godina bavimo sintezom N-aril supstituiranih 3, 4-HP s različitim supstituentima u para i meta položaju arilnog prstena. Daljnje strukturne modifikacije temeljnih piridinonskih struktura usmjerene su ka: a) uvođenju manozne podjedinice u svrhu ispitivanja antiadhezijskog učinka testom inhibicije hemaglutinacije[3] i b) uvođenju lipofilne podjedinice, primjerice adamantanske, u svrhu ispitivanja antitumorskog učinka. U ovom radu opisana je priprava novih adamantanskih i manoznih derivata 3, 4-HP. Temeljne piridinonske strukture s p- aminofenilom i p-hidroksifenilom skupinom na dušikovom atomu nisu bile pogodne za regioselektivno vezanje adamantanske i manozne podjedinice na hidroksilnu skupinu na položaju 3. Stoga je za pripravu p-aminofenilnih derivata korišten 3, 4-HP s p-nitrofenilnim supstituentom na dušikovom atomu, dok je za pripravu p- hidroksifenilnih derivata korišten 3, 4-HP s acetilom zaštićenom hidroksilnom skupinom u para položaju N-arilnog dijela molekule.

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).

Kojic acid can be produced from various carbohydrate sources in an aerobic condition by a variety of microorganisms.1 It contains a polyfunctional heterocyclic skeleton (Fig. 1) with several important reaction centres enabling additional reactions and modifications (alkylation, acylation, a ring opening of the molecule, chelation, etc.). It has been shown that kojic acid and its derivatives possess anti-inflammatory, antifungal and antitumour activity as well as metal ion chelating ability.1 Based on our ongoing study of adamantylated2 and mannosylated3 N-aryl substituted pyridin-4- one derivatives which were synthesized for the purpose of investigating their potential antitumor and hemagglutination properties, respectively, we aim to design new heterocyclic kojic acid derivatives in order to test their biological activities as well. The structure of kojic acid will be modified in several ways: the introduction of adamantyl or α- mannopyranoside unit, ring opening and introduction of nitrogen in the heterocyclic structure. In this work we report the preparation of acylated kojic acid derivatives where adamant- 1-ylacetyl was used as acyl part. The incorporation of adamantyl unit was carried out using Steglich esterification method on both hydroxyl functionalities or selectively on either one by the aid of protecting group tehniques. The prepared compounds will be tested for their in vitro antiproliferative abilities on several human cancer cell lines. We believe that the future studies on biological properties of kojic acid derivatives may lead to the development of a new class of specific and effective pharmaceutical agents. 1. J. Brtko, L. Rondahl, M. Ficková , D. Hudecová, V. Eybl, M. Uher, Cent. Eur. J. Publ. Health, 12 (2004) S16–S18. 2. V. Petrović Peroković, B. Prugovečki, Ž Car, Croat. Chem. Acta 86 (2013) 317-323. 3. Ž. Car, T. Hrenar, V. Petrović Peroković, R. Ribić, M. Seničar, S. Tomić, Chem. Biol. Drug Des. 84 (2014) 393-401.

Derivati piridin-4-ona dobri su kompleksirajući reagensi što ih čini potencijalnim terapeuticima kao i reagensima pogodnim za ekstrakciju i spektrofotometrijsko određivanje metalnih iona. U biološkim sustavima vanadij je često prisutan u koordinacijskim kompleksima, pa proučavanje njegovih interakcija s piridinonima doprinosi razumijevanju mehanizama akumulacijskih procesa vanadija u organizmu. U ovom radu opisana je priprava 3-hidroksi-1- (p-hidroksifenil)-2-metilpiridin-4-ona (HH), u jednom koraku iz polaznog maltola (3-hidroksi- 2-metilpiran-4-ona) i p-hidroksianilina. Sintetiziranom piridinonskom derivatu određene su protonacijske konstante ravnoteže te molekulska i kristalna struktura. Spoj kristalizira u monoklinskom sustavu, u prostornoj grupi P21/n. U kristalnoj strukturi molekule se povezuju O1–H1 O2(-x, -y, -z) (d(O1 O2=2.6733(12) Å) i O3–H3 O2(3/2+x, 1/2-y, 1/2+z) (d(O3 O2=2.6733(12) Å) vodikovim vezama tvoreći neprekinutu 2D mrežu. Unutar molekule prisutne su O1–H1 O2 (d(O1 O2=2.7656(12) Å) vodikove veze. Spektrofotometrijskim mjerenjima ispitana je reakcija HH i vanadija(V). U kiseloj vodenoj otopini nastaje ružičasti kompleks s maksimumom apsorpcije pri 507 nm u kojem je odnos V(V) : HH = 1 : 2. Intenzitet apsorpcije povećava dodatak etanola. Nastali V(V)–HH kompleks može se ekstrahirati u kloroform uz hipsokromni pomak apsorpcijskog maksimuma.

We investigated efficiency of novel imidazolium and benzimidazolium oximes to reactivate organophosphorus compounds paraoxon‐, VX‐, and tabun‐inhibited cholinesterases (ChEs). Their antidotal potency, expressed in terms of the observed reactivation rate constant (kobs ), was compared with the potency of HI‐6 and obidoxime, used in medical practice today. None of the tested oximes showed overall improvement in the reactivation of OP‐inhibited AChE, however, several oximes were pointed out as leads in the case of OP‐inhibited BChE reactivation. A comprehensive analysis was performed for the most promising oximes defining all relevant kinetic parameters of reactivation as well as interactions with uninhibited ChEs. Considering the strict criteria set for in vivo applications, we determined cytotoxicity of lead oximes on two cell lines, as well as ex vivo bioscavenging capacities of BChE‐oxime pairs. Furthermore, experimental data were correlated with computational studies including QSAR analysis as a starting point in further oximes’ structure refinement. Obtained overall profiles of lead oximes allow potential in vivo tests in development of BChE based OP‐ bioscavengers. Supported by the CSF 4307.

Muropeptidi su fragmenti peptidoglikana, polimera koji izgrađuju stanične stijenke bakterija. Koriste se kao imunostimulatori (adjuvanti). Muramildipeptid (MDP), N-acetilmuramil-L-alanil-D- izoglutamin, je najmanja strukturna podjedinica peptidoglikana koja pokazuje adjuvantsko djelovanje. Važan parametar na koji treba utjecati, s ciljem da se poboljšaju farmakološka svojstva MDP-a, jest lipofilnost. Stoga su pripravljeni brojni MDP analozi bez hidrofilne šećerne podjedinice (desmuramildipeptidi) te derivati s različitim skupinama vezanim na C- i N- kraju dipeptida. Mi smo sintetizirali desmuramilpeptide s adamantanskom podjedinicom te njihove manozne derivate. Manoza je uvedena kako bi se omogućila interakcija sa specifičnim lektinima, kao što su manozni receptori (MR), odnosno omogućila ciljana dostava spoja do stanica s eksprimiranim MR-ovima. Peptidi s vezanim adamantanom i manozom ugrađeni su u liposome, na način da adamantan ugradi u lipidni dvosloj, a manoza ostaje izložena na površini liposoma i služi kao meta za MR-ove. Nadalje, vezanjem manoze na muropeptide utjecalo se na smjer imunorekcije.

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).