SHORT COMMUNICATION Design, synthesis and antimycobacterial evaluation of some new azaheterocycles with the 4,7-phenanthroline skeleton. Part VI J. Serb. Chem. Soc. 81 (2) 133–140 (2016) UDC 547.235.2’7+547.677.5:547.639.1’583.1: JSCS–4833 615.281/.282 Short communication 133 SHORT COMMUNICATION Design, synthesis and antimycobacterial evaluation of some new azaheterocycles with the 4,7-phenanthroline skeleton. Part VI CRISTINA M. AL MATARNEH1, CATALINA I. CIOBANU2, IONEL I. MANGALAGIU1 and RAMONA DANAC1* 1Alexandru Ioan Cuza University of Iasi, Faculty of Chemistry, Chemistry Department, 11 Carol I, Iasi 700506, Romania and 2Alexandru Ioan Cuza University of Iasi, Faculty of Chemistry, Research Department, 11 Carol I, Iasi 700506, Romania (Received 14 May, revised 31 August, accepted 5 October 2015) Abstract: A feasibility study concerning the synthesis, structure and in vitro antimycobacterial evaluation of new 4,7-phenanthroline derivatives is reported. The preparation is straightforward and efficient, involving an N-alkylation reaction of 4,7-phenanthroline. The structure of the new compounds were verified by elemental and spectral (IR, 1H- and 13C-NMR) analysis. The in vitro antimycobacterial evaluation of the five synthesized compounds was investigated against Mycobacterium tuberculosis H37Rv under aerobic con- ditions. A certain influence of substituents on the para position of the benzoyl moiety was observed; the 4,7-phenanthrolin-4-ium salt substituted with p-chlorobenzoyl group showing the most pronounced antimycobacterial activity. Keywords: p-halogenobenzoyl; cycloimmonium salts; antimycobacterial; 4,7-phenanthroline; N-alkylation. INTRODUCTION Phenanthroline derivatives have attracted attention especially due to their biological effects,1–6 crystal engineering,7–11 unique π-electrons delocaliz- ation12,13 and complexation properties, especially in the case of 1,10-phenan- throline.14,15 While 1,10-phenathroline derivatives have been widely studied both for synthesis and applications, much less interest has been shown for the other phenanthrolines because of difficulties in their synthesis. However, there are several reports regarding biological properties of 4,7- -phenanthroline and its derivatives, such as inhibition of several enzymes,16–18 microbicide activity especially as an amoebicide19 and antiviral activity.20 More * Corresponding author. E-mail: rdanac@uaic.ro doi: 10.2298/JSC150514084A _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 134 AL MATARNEH et al. recently, 4,7-phenanthroline derivatives were found to stabilize triple-helix DNA21 and to possess in vitro and in silico antiviral activity against single- stranded positive-sense RNA genome viruses.22 As part of ongoing research in the field of heterocyclic compounds, espe- cially in the synthesis of (aza)indolizine and poly(aza)indolizine derivatives via (3+2) cycloaddition of cycloimmonium ylides,23–25 and encouraged by previous promising results in the field of anti-TB derivatives with a nitrogen heterocycle skeleton,26–30 it was decided to study the synthesis, structure and in vitro anti- mycobacterial activity of new 4,7-phenanthrolin-4-ium salts. EXPERIMENTAL Chemistry Melting points were recorded on an A. Krüss Optronic melting point meter KSPI and are uncorrected. Proton and carbon nuclear magnetic resonance (δH, δC) spectra were recorded on a DRX-500 Bruker (500 MHz) instrument. All chemical shifts are quoted on the δ-scale in ppm. Coupling constants are given in Hz. IR spectra were recorded on a Shimadzu Prestige 8400S FTIR spectrophotometer. All commercially available products were used without further purification unless otherwise specified. General procedure for the synthesis of 4,7-phenanthrolin-4-ium salts (1–8) 4,7-Phenanthroline (28 mmol, 1 equiv.) was dissolved in 6 mL anhydrous acetonitrile. Then a reactive halide 30.8 mmol (1.1 equiv.) was added and the resulting mixture was stirred at reflux for 24 h. The formed precipitate was filtered and washed with acetonitrile and diethyl ether to give the desired product. The following compounds were synthesized: 4-(2-oxo-2-p-tolylethyl)-4,7-phenanthrolin- 4-ium bromide (1), 4-(2-(4-methoxyphenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (2), 4-(2-(4-nitrophenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (3), 4-(2-(4-chloro- phenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (4), 4-(2-(3-methoxyphenyl)-2-oxo- ethyl)-4,7-phenanthrolin-4-ium bromide (5), 4-(2-amino-2-oxoethyl)-4,7-phenanthrolin-4-ium iodide (6), 4-(cyanomethyl)-4,7-phenanthrolin-4-ium bromide (7) and 4-(2-methoxy-2-oxo- ethyl)-4,7-phenanthrolin-4-ium bromide (8). The physical and spectral data for compounds 1–8 are given in the Supplementary material to this paper. Microbiology The compounds were evaluated for their antimycobacterial activity against Mycobac- terium tuberculosis, as a part of the TAACF TB screening program under direction of the US National Institute of Health, the NIAID division. The evaluation of the antimycobacterial activities of the compounds were performed at the Center of Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) of the Southern Research Institute.31-34 Primary cycle high throughput screening (HTS). Determination of the 90 % inhibitory concentration (IC90), 50 % inhibitory concentration (IC50) and minimum inhibitory concen- tration (MIC) The MIC values of the compounds were determined by measuring the bacterial growth after 5 days in the presence of test compounds. Compounds were prepared as 10-point two- -fold serial dilutions in DMSO and diluted into 7H9-Tw-OADC medium in 96-well plates with a final DMSO concentration of 2 %. The highest concentration of the compounds was _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS OF NEW 4,7-PHENANTHROLINE DERIVATIVES 135 200 µM when the compounds were soluble in DMSO at 10 mM. For compounds with limited solubility, the highest concentration was 50× less than the stock concentration, e.g., 100 µM for 5 mM DMSO stock and 20 µM for 1 mM DMSO stock. For potent compounds, the assays were repeated at lower starting concentrations. Each plate included assay controls for background (medium/DMSO only, no bacterial cells), zero growth (100 µM rifampicin) and maximum growth (DMSO only), as well as a rifampicin dose response curve. The plates were inoculated with M. tuberculosis and incubated for 5 days: growth was measured by the optical density at 590 nm (OD590) and fluorescence (Ex. 560 nm/Em. 590 nm) using a BioTek™ Synergy 4 plate reader. Growth was calculated separately for OD590 and relative fluorescence units (RFU). To calculate the MIC, the 10-point dose response curve was plotted as % growth and fitted to the Gompertz model using GraphPad Prism 5. The MIC was defined as the minimum concentration at which growth was completely inhibited and was calculated the Gompertz model from the inflection point of the fitted curve to the lower asymptote (zero growth, Fig. 1A). In addition, dose response curves were generated using the Levenberg– –Marquardt algorithm and the concentrations that resulted in 50 and 90 % inhibition of growth were determined (IC50 and IC90, respectively, Fig. 1B). Fig. 1. Dose response curves used to calculate the MIC, IC50 and IC90. MIC values were reported when the following quality control criteria were satisfied: – For each plate: no growth in the background (un-inoculated) control wells; OD590 >0.3 in maximum growth wells; rifampicin MIC within 3-fold of the expected value. – For each compound curve, the MICs were reported if there were 2 points with growth >75 %. If only one point was >75 % inhibition, then the MIC value was reported as the maximum concentration tested. If no point reached 75 % inhibition, the MIC was reported as >maximum concentration tested. RESULTS AND DISCUSSION Chemistry According with the goal of this study, it was decided to synthesize new phenanthroline derivatives: 4,7-phenanthrolinium monoquaternary salts 1–8 and 4,7-phenanthrolinium diquaternary salts 9–16. The strategies adopted for the con- _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 136 AL MATARNEH et al. struction of the phenanthroline derivatives was straightforward and efficient, involving an N-alkylation reaction of 4,7-phenanthroline, Scheme 1. Scheme 1. Reaction pathway to obtain the 4,7-phenanthrolin-4-ium salts. Unfortunately, it was not possible to synthesize compounds 9–16, under any of the employed conditions (ambient temperature or refluxing, content of 4,7- -phenanthroline. Thus, on varying the amount of the reactive halides from 1:2 to 1:7, and using different solvents, i.e,. acetone, acetonitrile and dimethylform- amide, only 4,7-phenanthrolin-4-ium mono salts 1–8 were obtained. As in the case of 1,7-phenanthroline,26 a feasible explanation for this behavior could be related to the basicity of the N7-nitrogen atom, i.e., after the N4-alkylation of 4,7-phenanthroline, the basicity of the N7-nitrogen is decreased in the obtained phenanthrolin-4-ium mono salts so that the second alkylation could not occur. The salts 1–8 were prepared in moderate to good yields (58–94 %), using a minimum volume of acetonitrile by refluxing the reaction mixture for 24 h. The structure of the new compounds was assigned by elemental and spectro- scopic analysis: IR, 1H- and 13C-NMR. The IR spectra of compounds 1–6 and 8 are characterized by intense absorption bands in the region of 1672–1698 cm–1 specific to C=O stretching, whereas in the IR spectrum of compound 7 the cyano group furnishes an absorption band at 2201 cm–1. In the 1H-NMR spectra of the new monoquaternary salts 1–5, the signals for methylene protons H15 appear at low fields (7.07–7.20 ppm, singlet), according to the substituent on the para or meta position of the benzoyl ring. The same protons appear more shielded at 5.27, 6.58 and 6.35 ppm for compounds 6–8, respectively, due to the weaker withdrawing effect of the adjacent carbonyl amide, cyano and ester groups. In the aromatic region, the most unshielded protons are H3 (the signals of which appear at 10.22–10.33 ppm) situated in the proximity of the positive nitrogen atom N4. In the 1H-NMR spectra of compound 6, the two-amide protons (NH2) furnished two singlet signals at 7.89 and 8.21 ppm, whereas in the spectrum of compound 8 _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS OF NEW 4,7-PHENANTHROLINE DERIVATIVES 137 the methyl ester protons appear as a singlet at 3.81 ppm. In the 13C-NMR spectra of compounds 1–5, the signals for C16 from the C=O ketone groups appear at 190.6–188.8 ppm, while for compound 6, having an amide group, and for com- pound 8 with an ester group, it appears at 165.8 and 166.6 ppm, respectively. The methylene C15 atoms give signals at 63.7–64.3 ppm for compounds 1–5 and at 45.6–59.5 ppm for compounds 6–8. All the other signals from NMR spectra are in agreement with the proposed structures. Design and biological activity In the continuing battle against M. tuberculosis, researchers have found that the 1,10-phenanthroline skeleton and a p-halogenobenzoyl moiety are useful pharmacophoric units for the antimycobacterial activity.35,36 Moreover, recent results26 in the area of 1,7-phenanthroline salts indicate that derivatives con- taining a p-substituted benzoyl moiety exhibit activity against M. tuberculosis H37Rv, the relative order of activity being p-Cl- > p-Br- > p-methyl-. Encouraged by these promising results in the field of anti-TB derivatives with the phenanthroline skeleton and, especially by recent results in the area of 1,7-phenanthroline salts which contain a p-substituted-benzoyl moiety, it was decided to combine the biological potentials of 4,7-phenanthroline and the p-sub- stituted benzoyl moiety, with the intention of obtaining compounds with better activity and better pharmacological properties, and to determine whether chang- ing the nitrogen atoms position of phenanthroline from 1,7- to 4,7- would some- how affect the activity, Scheme 2. Also in view were other 4,7-phenanthrolinium salts with the alkoxycarbonyl, cyano and acetamide moiety, in order to allow struc- ture–activity relationship (SAR) comparisons with p-substituted benzoyl salts. Scheme 2. Design in the class of 4,7-phenanthrolin-4-ium derivatives with p-halogeno benzoyl moiety. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 138 AL MATARNEH et al. A selection of compounds, salts 1–4 and 6, were evaluated for in vitro antimycobacterial activity against M. tuberculosis H37Rv (grown under aerobic conditions), as a part of ongoing collaboration with the TAACF TB screening program under direction of the US National Institute of Health, the NIAID division. The MIC value were determined by measuring bacterial growth after 5 days in the presence of the test compounds.30–34 The assay was based on mea- surement of the growth in liquid medium of a fluorescent reporter strain of H37Rv where the readout was either optical density (OD) or fluorescence. A linear relationship between the OD and fluorescence readout was established, justifying the use of fluorescence as a measure of bacterial growth. The MIC values generated from the OD measurements are reported in the summary data. The MIC was defined as the minimum concentration at which growth was com- pletely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth, Fig. 1A). In addition, dose response curves were generated using the Levenberg–Marquardt algorithm and the concentrations that resulted in 50 and 90 % inhibition of growth were determined (IC50 and IC90, respectively, Fig. 1B). The strain has been fully characterized and is equivalent to the parental strain in microbiological phenotypes and virulence. The obtained results are listed in Table I. TABLE I. Antimycobacterial activity of phenanthrolinium salts 1–4 and 6 against M. tuberculosis H37Rv under aerobic conditions Tested compound IC50 / μM IC90 / μM MIC / μM 1 (R=C6H4–Mep) 110 >200 >200 2 (R=C6H4–OMep) >200 >200 >200 3 (R=C6H4–NO2p) >200 >200 >200 4 (R=C6H4–Clp) 83 >200 >200 6 (R=NH2) >200 >200 >200 Rifampicin 0.0036 0.0061 0.0055 Although the results are not spectacular, the data from Table I illustrate that two of the five tested compounds exhibited activity against M. tuberculosis H37Rv: salt 4 (substituted with p-chlorobenzoyl moiety) and salt 1 (substituted with p-methylbenzoyl moiety), the first one having a more pronounced antimyco- bacterial activity. By comparison with similar 1,7-phenanthrolin-7-ium mono- quaternary salts it could be seen that the heterocycle has only a minor influence of on antimycobacterial activity, the IC50 values of the derivatives (1,7 or 4,7- -phenanthroline) substituted with the same substituents being similar, e.g., for 7-(2-(4-chlorophenyl)-2-oxoethyl)-1,7-phenthrolin-7-ium bromide, IC50 = 88 μM26, while for 7-(2-oxo-2-p-tolylethyl)-1,7-phenanthrolin-7-ium bromide, IC50 = = 100 μM.26 _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS OF NEW 4,7-PHENANTHROLINE DERIVATIVES 139 CONCLUSIONS The synthesis, structure and in vitro antimycobacterial activity of a new class of 4,7-phenanthrolin-4-ium monoquaternary halides are presented. The com- pounds were prepared by a straightforward and efficient method. The structure of the new compounds was assigned by elemental and spectroscopic analysis: IR, 1H-NMR and 13C-NMR. The in vitro antimycobacterial activity of the syn- thesized compounds was investigated against M. tuberculosis H37Rv under aero- bic conditions. Two of the five tested compounds showed activity against M. tuberculosis H37Rv, the 4,7-phenanthrolin-7-ium derivative substituted with p-chloro-benzoyl moiety being the most active. SUPPLEMENTARY MATERIAL The physical and spectral data for the prepared compounds are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. Authors C.M. Al Matarneh and C.I. Ciobanu are thankful to POSDRU/159/1.5/S/137750 for financial support. Part of this work (biological tests) was supported by National Institutes of Health and the National Institute of Allergy and Infectious Diseases, Contract No. HHSN272201100012I. We also thank the POSCCE-O 2.2.1, SMIS- -CSNR 13984-901, No. 257/28.09.2010 Project, CERNESIM, for the NMR results. И З В О Д ДИЗАЈН, СИНТЕЗА И ИСПИТИВАЊЕ АНТИМИКОБАКТЕРИЈСКЕ АКТИВНОСТИ НОВИХ АЗАХЕТЕРОЦИКЛА СА 4,7-ФЕНАНТРОЛИНСКИМ СКЕЛЕТОМ. 6. ДЕО CRISTINA M. AL MATARNEH1, CATALINA I. CIOBANU2, IONEL I. MANGALAGIU1 и RAMONA DANAC1 1Alexandru Ioan Cuza University of Iasi, Faculty of Chemistry, Chemistry Department, 11 Carol I, Iasi 700506, Romania и 2 Alexandru Ioan Cuza University of Iasi, Faculty of Chemistry, Research Department, 11 Carol I, Iasi 700506, Romania Приказано је истраживање у ком је описана синтеза, одређивање структуре и in vitro испитивање антимикобактеријске активности нових деривата 4,7-фенантролина. Синтеза деривата је директна и ефикасна и укључује реакције N-алкиловања 4,7-фен- антролина. Структура добијених деривата утврђена је елементалном анализом и спек- троскопским методама (IR, 1H- и 13C-NMR). Антимикобактеријска in vitro активност пет синтетисаних једињења испитана је према Mycobacterium tuberculosis H37Rv под аероб- ним условима. Утврђено је да постоји утицај супституената у пара положају бензоил језгра и да 4,7-фенантролин-4-ијум со са p-хлорбензоил групом показује веома изра- жену антимикобактеријску активност. (Примљено 14. маја, ревидирано 31. августа, прихваћено 5. октобра 2015) REFERENCES 1. G. R. Newkome, K. J. Theriot, V. G. Gupta, F. R. Fronczek, G. R. Baker, J. Org. Chem. 54 (1989) 1766 2. S. V. Vasileva, M. V. Stupakova, I. I. Lobysheva, V. D. Mikoyan, A. F. Vanin, Biochemistry (Moscow) 66 (2001) 984 3. A. D. Yapi, M. Mustofa, A. Valentin, O. Chavignon, J. C. Teulade, M. Mallie, J. P. Chapat, Y. Blache, Chem. Pharm. Bull. 48 (2000) 1886 _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 140 AL MATARNEH et al. 4. C. 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Available on line at www.shd.org.rs/JSCS/ << /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. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice