{Synthesis of sulfonamides bearing 1,3,5-triarylpyrazoline and 4-thiazolidinone moieties as novel antimicrobial agents} J. Serb. Chem. Soc. 85 (2) 155–162 (2020) UDC 547.541.521+547.772.2’78:542.913: JSCS–5290 615.272’285 Original scientific paper 155 Synthesis of sulfonamides bearing 1,3,5-triarylpyrazoline and 4-thiazolidinone moieties as novel antimicrobial agents THI-DAN THACH1,2, THI TUONG-VI LE1, HUU THIEN-AN NGUYEN2-4, CHI-HIEN DANG2,4**, VAN-SU DANG5 and THANH-DANH NGUYEN3,4** 1Tra Vinh University, Tra Vinh City, Tra Vinh Province, Vietnam, 2Graduate University of Science and Technology, Vietnam, Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam, 3Institute of Research and Development, Duy Tan University, Da Nang City, Vietnam, 4Institute of Chemical Technology, Vietnam Academy of Science and Technology, 1 Mac Dinh Chi Street, District 1, Ho Chi Minh City, Vietnam and 5Department of Chemical Technology, Ho Chi Minh City University of Food Industry, Vietnam (Received 21 June 2018, revised 8 April 2019, accepted 13 June 2019) Abstract: Two series of sulfonamides were synthesized from 4-hydrazinylben- zenesulfonamide as the key starting material. 1,3,5-Triarylpyrazoline sulfon- amides (2a–i) were obtained by cyclocondensation of various chalcones in 53– –64 % yields, while 4-thiazolidinone derivatives (4a–e) were synthesized by cyclocondensation between mercaptoacetic acid and different phenylhydra- zones in 43–62 % yields. The synthesized compounds were characterized based on FTIR, 1H-NMR, 13C-NMR and HRMS data. The sulfonamides were evaluated for their in vitro antimicrobial activities against four bacterial strains (E. coli, P. aeruginosa, B. subtillis and S aureus), two filamentous fungal strains (A. niger and F. oxysporum) and two yeast strains (C. albicans and S. cerevisiae). Seven pyrazolines, 2a–c and 2e–h, exhibited significant inhibition of different microbial strains. Among them, compound 2b displayed good anti- fungal activity against A. niger (MIC value at 12.5 μg mL-1) over the reference drug. Keywords: synthesis; sulfonamide; pyrazoline; thiazolidinone; antimicrobial agents. INTRODUCTION Sulfonamides have been particularly considered as essential scaffolds for developing new medicines. The sulfonamides possess a wide range of biological activities such as anti-inflammatory,1 antidepressant,2 antimicrobial3,4 and anti- cancer.5 In bacterial cells, antibacterial sulfonamides act as competitive inhibitors of the enzyme, dihydropteroate synthase that is involved in folate synthesis.6 *,** Corresponding authors. E-mail: (*)dangchihien@gmail.com; (**)danh5463bd@yahoo.com https://doi.org/10.2298/JSC180621057T ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. 156 THACH et al. Several sulfonamide drugs developed from aromatic compounds have been used in antimicrobial therapeutic applications. For instance, sulfanilamide and sulfa- methoxazole (Fig. 1) are the simplest antimicrobial therapy drugs which were commonly used in treatments against Gram-positive and Gram-negative bacteria, as well as in the treatments of fungi and protozoa infections. These sulfonamides are selectively toxic to bacteria via blocking of the synthesis of folic acid.7,8 However, resistance to these sulfonamides has increased during past years because the susceptible pathogens might develop an ability to take up folic acid from their environment.9 Fig. 1. Structures of antimicrobial therapy sulfa drugs. Pyrazolines are one of the important heterocycles containing two nitrogen atoms in the five-membered ring. These derivatives have been found to possess a broad spectrum of pharmacological actions, such as antimicrobial,10,11 anticon- vulsant,12 anti-inflammatory13 and antiviral.14 Numerous studies have shown that pyrazoline derivatives possess interesting antimicrobial activity against many fungal and bacterial strains. For example, Zampieri et al.15 reported synthesized pyrazoline derivatives bearing imidazole moieties with high activity against two strains, Candida albicans and Mycobacterium tuberculosis H37Rv. In particular, 1,3,5-substituted pyrazolines were documented as potential antibacterial agents.16–18 Additionally, 4-thiazolidinones, derivatives of thiazolidine with a carbonyl group at the 4-position, are an important scaffold known to be asso- ciated with many biological applications including anticancer, antimicrobial, antiviral and anti-inflammatory.19–22 In connection with our ongoing research program involving the synthesis and biological evaluation on heterocycles,23–25 the biological importance of sulfonamides prompted us to synthesize derivatives bearing pyrazoline and 4-thiazolidinone moieties, as well as to evaluate their antimicrobial activity. EXPERIMENTAL Chemistry All the materials were purchased from Acros (Belgium) or Aldrich. The solvents were purchased from Fluka and used without further purification. Melting points were measured with an Electrothermal Model 9200 (UK). The IR spectra were recorded on a Bruker Equinox 55 IR spectrophotometer. The ESI-MS were taken on an Aligent 1100 LC-MSD instrument ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. SYNTHESIS OF ANTIMICROBIAL SULFONAMIDES 157 with an MS detector (mass range 100–2000 m/z, energy 3.5 kV, dried gas 8.0 L min-1). 1H- (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker Avance 500 NMR spectrometer using acetone-d6, CDCl3 and DMSO-d6 as solvents and tetramethylsilane (TMS) as internal standard. Chemical shifts are reported in δ relative to TMS. Analytical and spectral data are given in Supplementary material to this paper. General procedure for the synthesis of dihydro-1H-pyrazol-1-ylbenzenesulfonamides (2) A stirred solution of chalcones 1 (3 mmol) and 4-hydrazinylbenzenesulfonamide (699 mg; 3 mmol) in anhydrous methanol (30 mL) was refluxed for 12 h, monitoring by TLC. After completion of the reaction, the mixture was cooled overnight at 0 °C. The separated solid was filtered and washed with hexane (30 mL). The residue was purified by recrystal- lization from ethanol and dried under the vacuum to afford the pure compounds 2. General procedure for the synthesis of 4-thiazolidinone sulfonamides (4) A stirred mixture of phenylhydrazones (1 mmol) and mercaptoacetic acid (1 mL) were refluxed for 2 h. After completion of the reaction, monitored by TLC, ethyl acetate (10 mL) was added into the mixture. The organic residue was washed with NaHCO3 solution (3×20 mL), water and dried by anhydrous Na2SO4. Then the solvent was evaporated and the crude product purified by column chromatography on silica gel 60 (230–400 mesh, E Merck, Darmstadt, Germany) with n-hexane/ethyl acetate (30:70 volume ratio) as the eluent. Antimicrobial assay The antimicrobial activity and the minimal inhibitory concentration (MIC) of the syn- thesized compounds were determined by the broth dilution method in 96-well microtiter mic- roplates, as reported previously.28 Briefly, stock solutions of the synthesized compounds at concentration of 2 mg mL-1 were prepared by dissolving 2 mg of the test compound in 1 mL dimethyl sulfoxide (DMSO). The stock solutions were serially diluted in 1 mL of corresponding sterile media (Eugon Broth (Difco, USA) for bacteria and mycophil (Difco, USA) for yeast). to obtain concentrations of 12.5–50 μg mL-1 and inoculated with a suspension (100 μL) of the respective microorganism. Four bacterial strains including two Gram negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 10145) and two Gram positive bacteria (Bacillus subtillis ATCC 11774 and Staphylococcus aureus subsp. aureus ATCC 11632) were used for the evaluation of antibacterial activity. Two filamentous fungal strains (Aspergillus niger (ATCC 6275) and Fusarium oxysporum (ATCC 7601)) and two yeast strains (Candida albicans ATCC 7754 and Saccharomyces cere- visiae (VTCC–Y–62)) were used for evaluating the antifungal properties of the synthesized compounds. Tetracycline and streptomycin (Sigma) were used as positive controls of the tests with Gram negative and Gram positive, respectively, and nystatin (Sigma) was used as posi- tive control for the test of filamentous fungi and yeast. Pure DMSO was used as the negative control. The MIC value for a sample is expressed as the lowest concentration that inhibits the tested microbial growth. RESULTS AND DISCUSSION Chemistry In the present strategy, the series of triarylpyrazoline sulfonamides contains a benzensulfonamide ring attached to a five-membered pyrazoline ring at posit- ion 1 and two aryl rings bearing varied functional groups attached to the pyrazo- line ring at positions 3 and 5. These derivatives (2a–i) were synthesized in meth- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. 158 THACH et al. anol by cyclocondensation of 4-hydrazinylbenzenesulfonamide and the corres- ponding chalcones (1a–i), which were prepared from the appropriate aromatic aldehyde and the corresponding ketone in base catalyst/ethanol at room tempe- rature for 5 h (Scheme 1). The present results showed that reaction time of the cyclocondensation (12 h) was much shorter than that of the previous report (36 h)13 while the yields were similar (53–64 %). Scheme 1. Synthesis of 1,3,5-triarylpyrazoline sulfonamides. Reagents: i) EtOH, KOH aq. (2 %), rt, 4 h; ii) 4-hydrazinylbenzenesulfonamide, MeOH, reflux, 12 h. The structures of triarylpyrazolines were unambiguously characterized by IR, NMR and mass spectral data. The 1H-NMR spectra showed characteristic singlet around 4.63–6.98 ppm for two protons of the –SO2NH2 moiety in the structures. In addition, signals belonging to the aliphatic protons of the pyrazoline ring, Ha, Hb and Hx appeared at around 3.15–3.27, 3.87–4.09 and 5.33–5.83 ppm, respect- ively. The other signals are in complete agreement with the assigned structures. The synthetic route of 4-thiazolidinone sulfonamides is illustrated in Scheme 2. The hydrazone derivatives (3a–e) were prepared in good yields (63.0–74.0 %) via acid catalyzed condensation of the appropriate aromatic aldehyde and 4-hyd- razinylbenzenesulfonamide in methanol for 4 h. Cyclocondensation of the appro- priate phenylhydrazones (3a–e) with a large excess of mercaptoacetic acid afforded the respective 4-thiazolidinone sulfonamides (4a–e). The IR data of 4a–e showed bands at around 3270 and 1700 cm–1 assigned to NH and CO stretching, respectively. In 1H-NMR spectra, protons of amines including NH and NH2 moi- eties are characterized by singlets at around 8.59–8.70 ppm and 7.04–7.06 ppm, respectively while the appearance of signals around 3.74–3.82, 3.84–3.95 and Scheme 2. Synthesis of 4-thiazolidinone sulfonamides. Reagents: i) CH3COOH, methanol, reflux, 4 h; ii) HSCH2COOH, reflux, 2 h. ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. SYNTHESIS OF ANTIMICROBIAL SULFONAMIDES 159 5.83–5.90 ppm, confirmed aliphatic protons of 4-thiazolidinone ring (Ha, Hb and Hx, respectively). Antimicrobial activity The pyrazoline and 4-thiazolidinone derivatives were screened for their acti- vity against four bacterial strains, including two Gram negative (E. coli and P. aeruginosa) and two Gram positive (B. subtillis and S aureus) bacteria, two fila- mentous fungal strains (A. niger and F. oxysporum) and two yeast strains (C. albicans and S. cerevisiae). The samples as the standard references including tet- racycline and streptomycin were used for the tests of Gram negative and Gram positive bacteria, respectively while nystatin was used for test of fungi and yeast. The data in average MIC values are given in Table I. It indicated that most of the pyrazoline sulfonamides exhibited high activity against three bacterial strains, E. coli, B. subtillis and S. aureus, both tested fungal strains and a yeast, C. albi- cans, whereas the 4-thiazolidinone derivatives did not show antimicrobial acti- vity at the tested concentration (50 μg mL–1). TABLE I. Antimicrobial activity (MIC / μg mL-1) of sulfonamide derivatives; T – tetracyc- line; S – streptomycin; N – nystatin Compd. Microorganism Bacterial Gram (–) Bacterial Gram (+) Fungi Yeast E. coli P. aerugi- nosa B. sub- tillis S. aur- eus A. niger F. oxyspo- rum S. cere- visiae C. albi- cans 2a >50 >50 >50 25 25 25 >50 >50 2b >50 >50 12.5 12.5 12.5 50 >50 >50 2c 50 >50 25 25 >50 >50 >50 25 2d >50 >50 >50 >50 >50 >50 >50 >50 2e 25 >50 25 25 >50 >50 >50 50 2f >50 >50 50 25 >50 >50 >50 >50 2g >50 >50 >50 >50 25 >50 >50 >50 2h >50 >50 >50 >50 25 >50 >50 >50 2i >50 >50 >50 >50 >50 >50 >50 >50 4a >50 >50 >50 >50 >50 >50 >50 >50 4b >50 >50 >50 >50 >50 >50 >50 >50 4c >50 >50 >50 >50 >50 >50 >50 >50 4d >50 >50 >50 >50 >50 >50 >50 >50 4e >50 >50 >50 >50 >50 >50 >50 >50 T 5.5 11.0 – – – – – – S – – 7.2 11.4 – – – – N – – – – 23.1 11.6 5.8 11.6 Amongst the synthesized pyrazolines, compound 2b having 2-hydroxy sub- stitution on benzene ring exhibited similar antibacterial activity to both Gram positive bacteria (MIC values at 12.5 μg mL–1) in comparison with streptomycin and superior antifungal activity to A. niger (MIC value at 12.5 μg mL–1) in res- ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. 160 THACH et al. pect to nystatin (MIC value at 23.1 μg mL–1). It is noteworthy that only derivat- ives 2c and 2e containing 4-tolyl substitut attached to the pyrazoline ring at position 5 showed activity against E. coli (MIC values at 50 and 25 μg mL–1, res- pectively) and yeast, C. albicans (MIC values at 25 and 50 μg mL–1, respect- ively) while three pyrazoline compounds 2g, 2h and 2i bearing 4-methoxyphenyl substituent at position 3 did not show antibacterial activity at the tested concen- tration. The results reveal that substituents in the structure of triarylpyrazoline sulfonamides have important effects on the antimicrobial activity. In general, a 4-methylphenyl substituent attached to pyrazoline ring exhibited activity over the 4-methoxyphenyl substituent and the pyrazolines containing hydroxy group can be important antibacterial agents. It is essential for the next research to prepare new antimicrobial triarylpyrazoline sulfonamides with excellent activity. CONCLUSIONS In summary, 1,3,5-triarylpyrazoline and 4-thiazolidinone sulfonamides were prepared from 4-hydrazinylbenzenesulfonamide in good yields. All the syn- thesized sulfonamides including novel compounds 2f, 2h, 4c and 4d, were well characterized by spectral analyses and investigated for their antimicrobial acti- vity. Seven pyrazoline sulfonamides 2a–c and 2e–h exhibited high activity against the different strains, whereas the 4-thiazolidinone derivatives did not dis- play antimicrobial activity at the tested concentration. Among them, compound 2b was found to be a potential antibacterial and antifungal agent compared with the references, streptomycin and nystatin, respectively. SUPPLEMENTARY MATERIAL Experimental data for synthesis of compounds 1a–i and 3a–e, NMR and HRMS spectra of compounds 2 and 4 are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. The authors are thankful to the Tra Vinh University, Tra Vinh City for financial assistance (No. 243/HĐ.ĐHTV-KHCN). И З В О Д СИНТЕЗА НОВИХ ДЕРИВАТА СУЛФОНАМИДА КОЈИ САДРЖЕ 1,3,5-ТРИАРИЛПИРАЗОЛИНСКЕ И 4-ТИАЗОЛИДИНОНСКЕ СТРУКТУРНЕ ДЕЛОВЕ, КАО АНТИМИКРОБНИХ АГЕНАСА THI-DAN THACH1,2, THI TUONG-VI LE1, HUU THIEN-AN NGUYEN2-4, CHI-HIEN DANG2,4, VAN-SU DANG5 и THANH-DANH NGUYEN3,4 1 Tra Vinh University, Tra Vinh City, Tra Vinh Province, Vietnam, 2 Graduate University of Science and Technology, Vietnam, Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam, 3 Institute of Research and Develop- ment, Duy Tan University, Da Nang City, Vietnam, 4 Institute of Chemical Technology, Vietnam Academy of Science and Technology, 1 Mac Dinh Chi Street, District 1, Ho Chi Minh City, Vietnam и 5 Department of Chemical Technology, Ho Chi Minh City University of Food Industry, Vietnam Две серије сулфонамида су синтетисане полазећи од 4-хидразинилбензенсулфонамида као полазног једињења. 1,3,5-Триарилпиразолин-сулфонамиди (2a–i) добијени су реакцијом ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 SCS. SYNTHESIS OF ANTIMICROBIAL SULFONAMIDES 161 циклокондензације различитих халкона у приносу 53–64 %, док су 4-тиазолидинонски дери- вати (4a–e) добијени реакцијом циклокондензације 2-меркаптосирћетне киселине са разли- читим фенилхидразонима у приносу 43–62 %. Синтетисана једињења су окарактерисана FTIR, 1H- и 13C-NMR и масеним спектрима. Испитана је in vitro антимикробна активност према четири бактерије (E. coli, P. aeruginosa, B. subtillis и S. aureus), два филаментна соја гљивица (A. niger и F. oxysporum) и два соја квасца (C. albicans и S. cerevisiae). Седам пира- золина 2a–c и 2e–h показују значајну инхибицију различитих микробних сојева. Од њих, једињење 2b показује добру антифунгалну активност према гљивици A. niger (MIC 12,5 μg mL-1), у поређењу са референтним леком. (Примљено 21. јуна 2018, ревидирано 8. априла 2019, прихваћено 13. јуна 2019) REFERENCES 1. N. V. Chandrasekharan, H. Dai, K. L. Roos, N. K. Evanson, J. Tomsik, T. S. Elton, D. L. Simmons, Proc. Natl. Acad. Sci. 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Oil Res. 30 (2018) 285 (https://doi.org/10.1080/10412905.2018.1435428). ________________________________________________________________________________________________________________________Available on line at www.shd.org.rs/JSCS/ (CC) 2020 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. 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