{Synthesis of novel piperazino-alkyl-1H-benzo[d]imidazole derivates and assessment of their interactions with the D2 dopamine receptor} J. Serb. Chem. Soc. 84 (9) 925–934 (2019) UDC 547.861.3’53.024+547.233– JSCS–5235 304.2:539.196:615 Original scientific paper 925 Synthesis of novel 2-(piperazino-1-yl-alkyl)-1H-benzimidazole derivates and assessment of their interactions with the D2 dopamine receptor JELENA Z. PENJIŠEVIĆ1, DEANA B. ANDRIĆ2, VLADIMIR B. ŠUKALOVIĆ1, GORAN M. ROGLIĆ2#, VUKIĆ ŠOŠKIĆ3 and SLAĐANA V. KOSTIĆ-RAJAČIĆ1* 1ICTM-Department of Chemistry, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia, 2Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia and 3Orgentec GmbH, Carl-Zeiss-Str. 49, 55129 Mainz, Germany (Received 29 October, revised 3 December, accepted 4 December 2018) Abstract: A total of 14 novel arylpiperazines were synthesized, and pharmaco- logically evaluated by measuring their affinities towards the D2 dopamine receptor (DRD2) in a [3H]spiperone competition assay. All the herein described compounds consist of a benzimidazole moiety connected to N-(2-methoxyphenyl)piperazine via linkers of various lengths. Molecular docking analysis and molecular dynamics simulations were performed on the DRD2–arylpiperazine complexes with the objective of exploring the receptor–ligand interactions and properties of the rec- eptor binding site. The recently published crystal structure of DRD2 was used throughout this study. The major finding is that high affinity arylpiperazines must interact with both the orthosteric binding site and the extended binding pocket of DRD2 and therefore should contain a linker of 5 or 6 methylene groups long. Keywords: arylpiperazines; molecular dynamics; molecular docking; receptor binding site. INTRODUCTION Dopamine receptors belong to the rhodopsin-like, aminergic G protein- -coupled receptors (GPCRs) group. They are involved in many physiological processes and play important role in the central nervous system (CNS).1–4 Targeting the dopamine D2 receptors (DRD2) is a common strategy for the treatment of neurodegenerative diseases, such as schizophrenia, Parkinson’s disease, dementia and depression.5–8 It is a well-documented fact that N-substituted arylpiperazines are com- pounds with pronounced DRD2 activity.9,10 Since arylpiperazines have a wide * Corresponding author. E-mail: srkostic@chem.bg.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC181029104P ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 926 PENJIŠEVIĆ et al. spectrum of therapeutic potentials and the design, synthesis and characterization of new arylpiperazine like drugs is an ever growing field of research.11–14 In this paper, the synthesis of 14 new N-(2-methoxyphenyl)piperazines of the general structure 5 (Scheme 1) is presented. Their affinities towards DRD2 were evaluated in the [3H]spiperone competition assay. Recent discovery of DRD2 crystal structure with bound risperidone15 def- ined the receptor binding site with greater accuracy than existing homology models. This finding prompted us to investigate DRD2–arylpiperazine binding features, using molecular docking analysis and molecular dynamics simulations in order to define key receptor–ligand interactions and explain the dopaminergic properties of the herein described compounds. EXPERIMENTAL The reagents and solvents used in this work were obtained from Alfa–Aesar or Sigma– –Aldrich and used without further purification. Solvents were routinely dried over anhydrous Na2SO4 prior to evaporation. General A Boetius PHMK apparatus (VEB Analytic, Dresden, Germany) was used to determine the melting points, which are here presented uncorrected. The 1H-NMR and 13C-NMR spectra were recorded at 200 and 50 MHz, respectively, on a Gemini 2000 (Varian, Oxford). The spectra were recorded in deuterochloroform with tetramethylsilane as the internal standard; the chemical shifts (δ) are reported in parts per million (ppm); all coupling constants (J values) are reported in Hz. LC/MS was performed on a 6210 time-of-flight LC–MS system (Agilent Technologies, Germany). For data analysis, MassHunter workstation software was used. The infrared (IR) spectra were obtained on a Thermo Scientific spectrometer. For ana- lytical thin-layer chromatography (TLC), Polygram SIL G/UV254 plastic-backed thin layer silica gel plates were used (Macherey–Nagel, Germany). The chromatographic purifications were performed on Merck-60 silica gel columns (230–400 mesh ASTM) under medium pres- sure (dry column flash chromatography). Analytical and spectral data for the synthesized compounds are given in Supplementary material to this paper. A MicroSYNTH Milestone and a Biotage Initiator 2.5 EXP were used for the microwave experiments. Chemistry General procedure for the synthesis of compounds 3a–g. A suspension of 1-(2-methoxy- phenyl)piperazine (1, 0.084 mol), triethylamine (0.0874 mol), K2CO3 (0.175 mol) and bromo- ester 2a–g (0.084 mol) in 2-butanone (100 mL) was stirred for 24 h at 80 °C. After cooling, the mixture was poured into cold water and the organic layer was extracted with CH2Cl2 and concentrated in vacuo. The resulting ester was purified by silica gel column chromatography using a gradient of methanol (0–5 %) in dichloromethane. General procedure for the synthesis of compounds 5a–n. Compounds 3a–g (0.0035 mol) and diamines 4a–c (0.0035 mol) were suspended in 8 mL 50 % methanesulfonic acid in water, transferred into a sealed tube, and microwave irradiated at 180 °C for 45 min at 300 W. After cooling to room temperature, the reaction mixture was poured into ice-cold water and neutralized with a saturated solution of NaOH. The product was extracted with CH2Cl2 and concentrated in vacuo. The resulting 1H-benzimidazoles were purified by silica gel column chromatography using a gradient of methanol (0–5 %) in dichloromethane. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. INVESTIGATION OF SYNTHESIZED DOPAMINERGIC LIGANDS 927 Biological assays Membrane preparation. Rat caudate nuclei synaptosomal membranes for the DRD2 binding experiments were prepared as previously described.16 Striatal tissue acquired from male Wistar rats (150–200 g) was used as the source of DRD2. The tissue was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 2 mM CaCl2 using a Potter–Elvehjem homogenizer (6×800 rpm). The mem- brane fraction obtained after centrifugation at 20000 rpm for 15 min was used in the binding experiments. [3H]Spiperone receptor binding assay. [3H]Spiperone (73.36 Ci mmol-1, Perkin Elmer LAS GmbH, Rodgau, Germany) binding was assayed in 1.0 mM EDTA, 4 mM MgCl2, 1.5 mM CaCl2, 5 mM KCl, 120 mM NaCl, 25 mM Tris-HCl solution, pH 7.4, with rat caudate nuclei synaptosomal membranes (protein concentration 0.6 mg mL-1), at 37 °C for 10 min in a total volume of incubation mixture of 0.4 mL. The binding of the radioligand to 5-HT2 rec- eptors was prevented by 50 mM ketanserin. The Ki values of the tested compounds were det- ermined by competition binding at 0.2 nM of the radioligand and eight different concen- trations of each compound (10-4–10-10 M). Nonspecific binding was determined in the pre- sence of 10 µM spiperone. The reaction was terminated by rapid filtration through Whatman GF/C filters, washed three times with 5.0 mL of ice-cold incubation buffer, and the retained radioactivity was measured in a 1219 Rackbeta Wallac scintillation counter (EG&G Wallac, Turku, Finland). Inhibition curve construction and statistical (Student’s t-test) analysis were performed by Graph-Pad Prism (GraphPad Software Inc). Hill slope coefficients were fixed to unity during the calculations. Computational study Docking simulations. The docking procedure was performed using Forecaster software.17 The receptor model PDB code 6CM418 was used together with 2D structures of the ligands, prepared in ChemDraw.19 All structures were prepared in the software using default proce- dures. Rigid receptor, flexible ligand docking was carried out. The obtained docking struc- tures were examined and structures with the maximum number of receptor–ligand interactions were selected for further analysis. Binding poses metadynamics. The docking pose quality was assessed in terms of the fluctuations of the ligand RMSD (the root-mean-square deviation of atomic positions), and the persistence of important contacts between the ligand and the receptor (Metadynamics Binding PoseScore and Metadynamics Binding Persistence) using Desmond software and default para- meters.20 One docking pose with the lowest RMSD and best overall score was selected for molecular dynamics (MD) simulations. Construction of a protein–membrane system for molecular dynamics. The protein pro- tonation state was adjusted using the Schrodinger Protein Preparation module, at physiological pH (pH 7.4). The prepared protein was embedded into a POPC membrane bilayer using the Desmond system builder module,20 and oriented according to data from the Orientations of Pro- teins in Membranes (OPM) server.21 The embedded protein was solvated with TIP3P explicit water model, and the system was neutralized via counter ions and a salt solution of 0.15 M KCl. In this way, systems were obtained that were subjected to membrane relaxation protocol.20 MD simulations. Molecular dynamics (MD) simulations of the system were performed using Schrodinger Desmond software packages.20 OPLS 2003 forcefield22 was used to calcul- ate the interactions between all the atoms. For the calculation of long-range coulombic inter- actions, the particle–mesh Ewald (PME) method was used, with a cut-off radius of 9 Å for short-range van der Waals (vdW) and electrostatic interactions. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 928 PENJIŠEVIĆ et al. During the course of the simulation, a constant temperature of 310 K and a pressure of 1.01235 bar were maintained, using a Nose–Hoover thermostat,23 and the Martyna–Tobias– –Klein method.24 Time increments of 2.0 fs were used in the simulations. Finally, 100 ns MD simulation for the each ligand– DRD2 complex was performed and the collected trajectory frames used in the MD analysis to quantify the protein–ligand interactions. RESULTS AND DISCUSSION Compounds 5a–n were synthesized according to Scheme 1. The synthesis started with N-(2-methoxyphenyl)piperazine (1) that was alkylated with a series of homologous bromo-esters 2a–g, providing N-alkylated products 3a–g. Count- erpart diamines 4a–c were obtained by reduction of the corresponding 2-nitro precursors, using Raney-Ni and hydrazine hydrate under conditions described in earlier publications.25,26 Microwave assisted condensation of piperazines 3a–g and diamines 4a–c, under forcing, strongly acidic conditions, secured the desired benzimidazoles 5a–n. Scheme 1. Synthesis of the compounds 5a–n n = 1–7 for compounds 2a–g and 3a–g; ethyl esters of the general structure 2 were used in the synthesis of 3b, 3c, 3e and 3f; 4a (R = H); 4b (R= OMe); 4c (R= Cl); structures 5a–n are presented in Table I. DRD2 binding affinities of compounds 5a–5n were evaluated in vitro using [3H]spiperone as a standard dopaminergic radioactive ligand (Table I).27 Molecular docking simulation of the herein described 2-{[4-(2-methoxyphe- nyl)piperazin-1-yl]alkyl}-1H-benzo[d]imidazoles on D2DR was performed on the D2DR crystal structure published recently by Wang et al.15 They reported that the benzisoxazole moiety of risperidone interact with D2DR through Cys1183.36, Thr1193.37, Ser1975.46, Phe1985.47, Phe3826.44, Phe3906.52 and Trp3866.48 in the orthosteric binding site (OBS). OBS of D2DR is defined by the amino acid side chains of helices III, V and VI and also harbour Asp1143.32. Asp1143.32 forms an essential salt-bridge with protonated piperidine nitrogen of risperidone molecule. In addition D2DR has a secondary binding pocket, ext- ended binding pocket (EBP), that encloses the tetrahydropyridopyrimidinone ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. INVESTIGATION OF SYNTHESIZED DOPAMINERGIC LIGANDS 929 moiety of risperidone. EBP is bordered by the extracellular part of TM VII con- sisting of an extracellular loop 1 (EL1) and the junction of helices I, II and VII.15 TАBLE I. Chemical structures and DRD2 binding constants of 2-{[4-(2-methoxyphenyl)pip- erazin-1-yl]alkyl}-1H-benzimidazoles (5a–n); DRD2 binding constants (Ki) were determined in a [3H]spiperone displacement assay. The values are the mean of three independent experiments realized in triplicate, performed at eight competing ligand concentrations Ligand n R Ki ± SEM /nM 5a 1 H >1000 5b 2 H >1000 5c 3 H >1000 5d 4 H >1000 5e 5 H 24±1 5f 6 H 16±2 5g 7 H >1000 5h 4 OCH3 124±5 5i 5 OCH3 12±3 5j 6 OCH3 76±8 5k 7 OCH3 >1000 5l 4 Cl 109±9 5m 5 Cl 25±3 5n 6 Cl 102±3 Molecular docking simulations on the binding of 2-{[4-(2-methoxyphenyl)- piperazin-1-yl]alkyl}-1H-benzimidazoles into the crystal structure of DRD2 show that the (2-methoxyphenyl)piperazine moiety occupies DRD2 OBS, and interacts with Asp1143.32, Cys1183.36, Trp3866.48 and Phe3906.52, while the benzimidazole part interacts with Leu942.64, Ile184EL2,Trp100EL1, Phe3896.51, Thr4127.39 and Tyr4087.35 in the EBP (Fig. 1). Compounds with optimal linker length (five or six methylene groups in the linker) allow the benzimidazole moiety to reach EBP and to interact with Leu942.64, Trp100EL1, Phe3896.51, Thr4127.39 and Tyr4087.35 (Fig. 2). Com- pounds with shorter linker (5a–d) do not reach into the EBP, while ligands with seven methylene groups in the linker (5g and 5k) are too long to fit optimally into the D2DR binding cleft and protrude into the extracellular space. These results are in agreement with experimental data: compound 5d (with a 4 methylene groups linker) has affinity of over 1000 nM, while compounds 5e and 5f (with 5 and 6 methylene groups linker, respectively) have 24 and 16 nM, respectively. Compound 5g shows a sharp drop in affinity because of the length of the linker, which cannot be accommodated in the DRD2 bind cleft. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 930 PENJIŠEVIĆ et al. Fig. 1. Docking of ligand 5i to DRD2 is presented. View of the interactions between the 3D model of the DRD2 binding site and ligand 5i. The images show only the key amino acid residues of the receptor binding pocket. Figures (side view–left and top view–right) show docking of 5i viewed from different angles. Binding site ligand accessible surface is shown in the top view. In series of compounds substituted with methoxy and chloro groups, the highest DRD2 affinity was obtained with compounds 5i and 5m. Linker with 5 methylene groups facilitates optimal positioning of substituted benzimidazole part in the receptor EBP (Fig. 1). Shorter linkers, as it is obvious in series 5h–k and 5l–n, lead to decrease in receptor affinity due to sub-optimal placement of benzimidazole part in regard to the interacting residues Trp100EL1 and Tyr4087.35. To test the stability of obtained docking poses, MD simulations of the DRD2 and selected ligands were performed on inactive receptor state for 100 ns for each ligand. Obtained trajectories were analyzed with focus on the residues that form OBS and EBP (Table S-I of the Supplementary material). Most of the receptor–ligand interactions in OBS, observed in molecular docking simulations, persisted for a significant portion of MD run (>20 % total simulation time). Compounds with significant DRD2 affinity (5e–f, 5h–j and 5l–n) had a salt bridge between the protonated piperazine nitrogen of the ligand and Asp1143.32 of DRD2 preserved for more than 79–84 % of the simulation time. Additional interactions in OBS are aromatic interaction with Cys1183.36 (32–75 % of the simulation time), and edge-to-face interactions with Trp3866.48 (76–98 % of the simulation time) and Phe3906.52 (20–49 % of the simulation time). In the EBP, significant interactions are aromatic interactions (edge-to-face type) with Trp100EL1, Phe3896.51 and Tyr4087.35. Compounds 5e, 5f, 5i and 5m form an additional hydrogen bond with Thr4127.39. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. INVESTIGATION OF SYNTHESIZED DOPAMINERGIC LIGANDS 931 Fig. 2. Results of docking simulations for ligand 5e (A), 5f (B), 5i (C) and 5m (D) are pre- sented. Schematic representation of the best docking pose for all ligands are provided. For clarity, only amino acid residues in close contact with ligands are shown. Solid lines represent aromatic, while dotted lines represent electrostatic interactions. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. 932 PENJIŠEVIĆ et al. CONCLUSIONS Molecular docking and MD simulation provide important information that explains how the receptor–ligand complexes are formed. High affinity 2-{[4-(2- -methoxyphenyl)piperazin-1-yl]alkyl}-1H-benzimidazoles must simultaneously occupy both OBS and EBP. To establish key interactions both in OBS (salt bridge formation and aro- matic interactions) and EBP (aromatic interactions and hydrogen bond form- ation), the ligands should have a linker of five or six methylene groups. Linker flexibility and substituent size in the benzimidazole moiety determine ligand positioning inside the EBP and brings it in close contact with Trp100EL1 and Tyr4087.35, which are key interacting residues. Additionally, as can be concluded from the results of molecular dynamics, the affinity of the arylpiperazine ligands benefit greatly from possible formation of interactions of the arylpiperazine part of ligands with Thr4127.39 in EBP. It is clear that both Trp100EL1 and Tyr4087.35 can form aromatic interactions and/or hydrogen bonds. To establish the exact nature of interactions in EBP, modification of presented ligands, in terms of target synthesis of the compounds which can strictly form only one of these interactions, represent a guideline for further investigation. SUPPLEMENTARY MATERIAL Analytical and spectral data for the synthesized compounds, as well as additional results, are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. This work was funded by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (Grant 172032). И З В О Д СИНТЕЗА НОВИХ ДЕРИВАТА 2-(ПИПЕРАЗИНО-1-ИЛ-АЛКИЛ)-1Н-БЕНЗИМИДАЗОЛА И ПРОУЧАВАЊЕ ИНТЕРАКЦИЈА СА Д2 ДОПАМИНСКИМ РЕЦЕПТОРОМ ЈЕЛЕНА З. ПЕЊИШЕВИЋ1, ДЕАНА Б. АНДРИЋ2, ВЛАДИМИР Б. ШУКАЛОВИЋ1, ГОРАН М. РОГЛИЋ2, ВУКИЋ ШОШКИЋ3 и СЛАЂАНА В. КОСТИЋ-РАЈАЧИЋ1 1ИХТМ-Центар за хемију, Универзитет у Београду, Његошева 12, 11000 Београд, 2Хемијски факултет, Универзитет у Београду, Студентски трг12–16, 11000 Београд и 3Orgentec GmbH, Carl-Zeiss-Str. 49, 55129 Mainz, Germany У овом раду је презентована синтеза 14 нових арилпиперазина и одређен је њихов афинитет везивања за Д2 допамински рецептор (DRD2) тестовима компетиције са [3H]спипероном. По својој хемијској структури ова једињења представљају супституисане бензимидазоле повезане са N-(2-метоксифенил)пиперазинским делом, линкерима разли- читих дужина. У циљу испитивања лиганд-рецептор интеракција и особина везивног места DRD2, урађена је докинг анализа новосинтетисаних једињења и симулација молекулске динамике, користећи кристалну структуру рецептора. 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Soc. 81 (2016) 347 (https://doi.org/10.2298/JSC1510). ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2019 SCS. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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