Synthesis of Ibuprofen Conjugated Molecular Transporter ...downloads.hindawi.com/journals/jchem/2017/4746158.pdf · ResearchArticle Synthesis of Ibuprofen Conjugated Molecular Transporter

Research ArticleSynthesis of Ibuprofen Conjugated Molecular TransporterCapable of Enhanced Brain Penetration

Goutam Biswas1 Wanil Kim2 Kyong-Tai Kim2 Junhwi Cho3

Dongjun Jeong4 Keon-Hyoung Song5 and Jungkyun Im3

1 India Innovation Research Centre 465 Patparganj Industrial Area Delhi 110092 India2Department of Life Science Division of Molecular and Life Science and Division of Integrative Biosciences and BiotechnologyPohang University of Science and Technology Pohang Republic of Korea3Department of Chemical Engineering Soonchunhyang University Asan Republic of Korea4Department of Pathology Soonchunhyang University Asan Republic of Korea5Department of Pharmaceutical Engineering Soonchunhyang University Asan Republic of Korea

Correspondence should be addressed to Jungkyun Im jkim5279schackr

Received 23 August 2016 Revised 28 November 2016 Accepted 29 November 2016 Published 16 January 2017

Academic Editor Josefina Pons

Copyright copy 2017 Goutam Biswas et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Based on the strong evidences between inflammation and neurodegeneration nonsteroidal anti-inflammatory drugs (NSAIDs)such as ibuprofen are considered as effective agents to reduce the risk of Alzheimerrsquos and Parkinsonrsquos disease However the clinicaluse of NSAIDs in these diseases is limited by low brain distribution In this study we had synthesized ibuprofen conjugate whichhas good brain penetration S-(+)-Ibuprofen was covalently attached to a molecular transporter having FITC and eight terminalguanidine groups This conjugate showed good cellular uptake property in live cells It was also injected into a mouse and thedistribution of the compoundwas examined in each organThe conjugatewaswell delivered tomouse brain indicating the conjugateis able to cross the blood-brain barrier Our novel synthetic ibuprofen conjugate will hopefully deliver other NSAIDs into brain andis therefore applicable to the neurodegenerative diseases treatment or prevention

1 Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are effec-tive for relieving pain and reducing inflammation These arenonselective inhibitors of two isoforms of cyclooxygenase(COX) cyclooxygenase-1 (COX-1) and cyclooxygenase-2(COX-2) and thereby inhibit the production of hormone-likelipid compounds such as prostaglandins and thromboxanewhich cause inflammation pain fever and platelet aggre-gation [1 2] Approximately 33 million Americans annuallyuse these compounds for the symptomatic relief of headacheosteoarthritis rheumatoid arthritis low back pain and mus-culoskeletal conditions Moreover many epidemiologicalstudies demonstrate that NSAIDs have a neuroprotectiveeffect against neurodegenerative diseases such asAlzheimerrsquosdisease and Parkinsonrsquos disease [3ndash7] Brain response tocellular stress injury and pathology can lead to neuroin-flammation which upregulatesmicroglia cellsThe aberrantly

activated microglia could induce chronic inflammation andcellular damage by producing diverse cytokines and reac-tive oxygen species (ROS) accelerating neuronal death andresulting in neurodegenerative disease [8ndash10] Since NSAIDscan reduce inflammation in brain and allow beneficial effectson preventing or reducing neurodegenerative diseases alongwith the principal therapeutic effect (eg anti-inflammation)of NSAID herein we would focus on its neuroprotectiveeffects

Ibuprofen is a propionic acid derivative among NSAIDclasses and was the first nonaspirin NSAID to be availableover the counter and has become the most commonly usedNSAID Recently it is reported that long term oral admin-istration of ibuprofen could delay the onset of Alzheimerrsquosas is found with other NSAIDs [11 12] When ibuprofen wasfed to transgenic mice models for Alzheimerrsquos both centralnervous system (CNS) inflammation and Alzheimer plaquedeposition in the brain were significantly reduced [11] In

HindawiJournal of ChemistryVolume 2017 Article ID 4746158 10 pageshttpsdoiorg10115520174746158

2 Journal of Chemistry

BBB BBB BBB

Blood

Brain

Ibuprofen

Ibuprofen-HOO

conjugate

Figure 1 The presence of BBB prevents the ibuprofen internaliza-tion into the brain Our objective is to make ibuprofen conjugatepass through the BBB

clinical research data from almost 250000 cases showedthose who took NSAID for more than five years were morethan 40 less likely to develop Alzheimerrsquos [12] The studyalso revealed that among the NSAIDs taken by patientsibuprofen showed the most profound effect Furthermore astudy suggests taking ibuprofen regularly to be also associatedwith lowering the risk of the development of Parkinsonrsquosdisease [13] The study reported that those taking ibuprofenregularly more than six years showed a 38 lower risk ofdeveloping the disorder than nonusers and other NSAIDssuch as aspirin and paracetamol did not show any effect tothe patients

However there are many challenges to be overcome foribuprofen and other NSAIDs to be used as treatments forneural disorders First the presence of blood-brain barrier(BBB) has been reported to significantly limit the brainpenetration of NSAIDs (Figure 1) [14ndash17] The BBB betweencerebral blood and the interstitial fluid of the brain actsas protection from an influx of pathogens as well as smalluseful molecular drugs like NSAIDs In addition ibuprofenis ionized at physiological pH and circulates as anions whichare extensively bound to plasma proteins by noncovalentassociations [18 19] As a consequence brain penetration ofibuprofen was found to be low in rats showing the total brainto plasma ratio being less than 002 Therefore ibuprofenreaching the human brain exists in only a tiny amount andhigh doses might be required to attain a certain level ofdrug efficacy toward neural disorders Also long term useof ibuprofen can cause a number of side-effects such ascardiovascular and gastrointestinal risks

Therefore to make ibuprofen a more potent drug toprevent or alleviate neurodegenerative diseases even in lowdoses delivery of ibuprofen to brain should be effective andibuprofen must penetrate the BBB and gain access into thepatientrsquos brain

We have recently developed several types of guanidine-richmolecular carrierswith highmolecularweights and goodwater solubility that readily cross the BBB anddisplay efficientdistributions in themouse brain [20 21] In particular the G8(having eight guanidine groups) molecular carrier based on

carbohydrate such as sorbitol was found to be very effectivein delivering conjugated drugs to mouse brain [22ndash25] Inthe present study employing the same molecular carrierwe prepared the ibuprofen conjugate and studied its BBBpermeability by mouse tissue distribution study

2 Experimental

21 Materials and Methods in Synthesis Analytical TLCwas performed on Merck 60 F254 silica gel plate (025mmthickness) and visualized under UV light andor by sprayingwith a 5 solution of phosphomolybdic acid (PMA) inethanol or ninhydrin specific for amino compounds followedby charring with a heat gun Column chromatography wasperformed on Merck 60 silica gel (70ndash230 or 230ndash400mesh)and MPLC on Fluka 100 C8-reversed phase silica gel NMRspectra were recorded on a Bruker ASPECT 300 instrumentoperating at 300MHz for 1H 75MHz for 13C Chemicalshifts were reported in parts per million (ppm) relative totetramethylsilane (TMS) or deuterium oxide (D2O) whichwere used as internal and external standard for the 1HNMRMass spectra were recorded on fast atom bombardment(FAB) conditions In case of highmass spectral dataMALDI-TOF were obtained on a Micromass MDI at the Biomolec-ular Diversity Core Facility (POSTECH) Analytical HPLCwas performed on Agilent 1100-HPLC Chemstation with ananalytical column ZORBAX C8-monomeric (BU-300 5 120583m300 A 46 times 250mm)

22 Characterization of Synthetic Compounds(S)-Ethyl-2-(2-(4-isobutylphenyl)propanamido)acetate (1) Asolution of (S)-(+)-ibuprofen (140mg 068mmol) glycineethylester hydrochloride (104mg 075mmol) EDC (195mg102mmol) triethylamine (104ml 0748mmol) and DMAP(42mg 034mmol) in DMF (2ml) was stirred at rt underN2(g) After stirring for 24 h the solution was treated withethylacetate and washed several times with saturated aqNaHCO3 water and brine The organic phase was driedover Na2SO4 and concentrated to give the crude productwhich was purified by column chromatography on silica geleluting with 1 5 EtOAchexane to afford (1) (199mg 99) asa colorless liquid119877119891 054 (Hexane EtOAc = 7 3) 1H-NMR (300MHz

CDCl3 ppm) 120575 099 (d J = 66Hz 6H) 123 (t J = 69Hz3H) 151 (d J = 72Hz 3H) 184 (m 1H) 244 (d J = 72Hz2H) 360 (q J = 72Hz 1H) 394 (dd J = 54Hz J = 126Hz2H) 415 (q J = 69Hz 2H) 711 (d J = 81Hz 2H) 722 (d J= 81Hz 2H) 13C-NMR (75MHz CDCl3 ppm) 120575 139 183222 300 413 449 462 611 1272 1294 1381 1405 16971745 MS (FAB) [M + H]+ calcd for C17H25NO3 mz 29218found 29220

(S)-2-(2-(4-Isobutylphenyl)propanamido)acetic acid (2)Com-pound (1) (195mg 067mmol) and lithiumhydroxide (16mg067mmol) were added to 6ml solution of THF H2O (5 1)and stirred at rt for 36 h Then the reaction mixture wasevaporated in vacuo extracted with EtOAc and washedwith saturated 1N HCl water and brine The organic phasewas dried over Na2SO4 and concentrated to give the crude

Journal of Chemistry 3

product which was purified by column chromatography onsilica gel elutingwith 1 2 EtOAchexane to afford (2) (105mg60) as a white solid119877119891 012 (Hexane EtOAc = 7 3) Mp 87ndash90∘C 1H-NMR

(300Mhz CDCl3 ppm) 120575 088 (d J = 66Hz 6H) 149 (d J= 72Hz 3H) 183 (m 1H) 243 (d J = 72Hz 2H) 362 (q J =72Hz 1H) 394 (dd J = 54Hz J = 183Hz 2H) 635 (m 1HNH) 709 (d J = 81Hz 2H) 718 (d J = 81Hz 2H)MS (FAB)[M + H]+ calcd for C15H21NO3 mz 26415 found 26417

1-O-(N-Cbz-6-aminohexanoyl)-2345-tetra-O-(N-bis-[3-(N1015840N10158401015840-bis-Boc-guanidino)-propyl]-6-aminohexanoyl)-6-O-d-sorbitol (4) A column of flash silica gel was packed con-secutively in hexane with 1 triethylamine and then hexanewith 1 TFA a sea sand layer was placed in between layersCompound (3) (98mg 0028mmol) was dissolved in 1TFAcontaining CH2Cl2 and sonicated for several seconds Thesolution was then loaded on the column chromatographyand was eluted with increasing MeOH in CH2Cl2 (15 1) togive compound (4) (66mg 73) as a colorless foamy solid119877119891 044 (CH2Cl2MeOH = 10 1) 1H-NMR (300MHz

CDCl3 ppm) 120575 122ndash179 (m 190H) 202ndash262 (m 32H) 315ndash348 (m 18H) 368 (m 2H) 431ndash440 (m 4H) 489ndash507(m 2H) 508 (s 2H) 510ndash530 (m 2H) 734 (m 5H) 851(brs 8H) 1148 (brs 8H) 13C-NMR (75MHz CDCl3 ppm)120575 141 227 240 243 248 253 263 271 280 281 283 283288 293 297 299 339 355 380 390 409 499 513 515515 531 636 665 792 797 800 805 830 833 837 845849 851 934 1145 1183 1226 1272 1279 1280 1285 12871367 1421 1433 1487 1530 1531 1558 1561 1567 16351718 1725 1727 1728 1731 1816 1850 1852

1-O-(2-Aminoacetoxy-ibuprofen)-2345-tetra-O-(N-bis-[3-(N1015840N10158401015840-bis-Boc-guanidino)-propyl]-6-aminohexanoyl)-6-O-(N-Cbz-6-aminohexanoyl)-d-sorbitol (5) To a solution of(4) (90mg 0027mmol) in DMF (2ml) at rt were added (2)(107mg 0041mmol) EDC (11mg 0054mmol) and DMAP(2mg 0016mmol) After stirring for 48 h under N2(g)The solution was concentrated under reduced pressure Theresiduewas dissolved inEtOAc andwashed several timeswithsaturated aq NaHCO3 water and brine The organic phasewas dried over Na2SO4 and concentrated to give the crudeproduct which was purified by column chromatographyon silica gel eluting with 1 20 MeOHCH2Cl2 to afford (5)(50mg 52) as a white foamy solid119877119891 046 (CH2Cl2MeOH = 10 1) 1H-NMR (300MHz

CDCl3 ppm) 120575 088ndash090 (m 6H) 125ndash169 (m 193H) 232ndash245 (m 32H) 308ndash350 (m 18H) 361ndash371 (m 2H) 397ndash429 (m 4H) 485ndash512 (m 4H) 502 (s 2H) 533 (m 1H)709ndash712 (m 2H) 722ndash727 (m 2H) 733 (m 5H) 849ndash857(m 8H) 1140 (m 8H) 13C-NMR (75MHz CDCl3 ppm) 120575141 186 224 227 240 243 246 263 280 281 283 283294 297 302 319 380 390 451 499 532 665 792 798830 837 1183 1226 1272 1274 1277 1279 1280 1285 12871295 1297 1367 1387 1405 1433 1530 1531 1562 15651568 1629 1635 1703 1726 1728 1743 1816

1-O-(2-Aminoacetoxy-ibuprofen)-2345-tetra-O-(N-bis-[3-(N1015840N10158401015840-bis-Boc-guanidino)-propyl]-6-aminohexanoyl)-6-O-d-sorbitol (6) A solution of (5) (45mg 0013mmol) in a mixed

solvent (CH2Cl2 MeOH = 1 9 10ml) was hydrogenated(50 psi) at rt over 10 PdC (25mg) After 12 h the catalystwas filtered and the filtrate was evaporated to give the aminocompound (6) (38mg 88) as a colorless sticky liquid1H-NMR (300 MHz CDCl3 ppm) 120575 088ndash090 (m 6H)

125ndash177 (m 193H) 225ndash245 (m 32H) 309ndash350 (m 18H)361ndash371 (m 2H) 401ndash429 (m 4H) 485ndash512 (m 4H) 533(m 1H) 709ndash712 (m 2H) 722ndash727 (m 2H) 856 (brs 8H)1139 (brs 8H)

1-O-(2-Aminoacetoxy-ibuprofen)-2345-tetra-O-(N-bis-[3-(N1015840N10158401015840-bis-Boc-guanidino)-propyl]-6-aminohexanoyl)-6-O-[6-(fluoresceinyl-5-thioureido)-hexanoyl]-d-sorbitol (7) Toa solution of amino compound (6) (38mg 0011mmol) ina mixed solvent THF and absolute ethanol (3ml 1 2) wereadded fluorescein-5-isothiocyanate (65mg 0017mmol)and triethylamine (46 120583l 0033mmol) The reaction mixturewas stirred for 36 h at rt in dark and then concentrated invacuo The crude product was purified on silica gel elutingwith 1 20 MeOHCH2Cl2 to afford (7) (25mg 60) as alight greenish-yellow sticky solid119877119891 042 (CH2Cl2 MeOH = 10 1) 1H-NMR (300MHz

CDCl3 ppm) 120575 088ndash090 (m 6H) 125ndash185 (m 193H) 231ndash244 (m 32H) 309ndash348 (m 18H) 366 (m 2H) 120575 415ndash429(m 4H) 488ndash507 (m 4H) 534 (m 1H) 651ndash732 (m 11H)773 (m 1H) 804 (m 1H) 857 (brs 8H) 1139 (brs 8H)

1-O-(2-Aminoacetoxy-ibuprofen)-2345-tetra-O-[N-bis-(3-guanidinopropyl)-6-aminohexanoyl]-6-O-[6-(fluoresceinyl-5-thioureido)-hexanoyl]-d-sorbitolsdot8HCl (8) To a solutionof (7) (20mg 0005mmol) in EtOAc (1ml) at rt was addedthe HCl(g) saturated solution of EtOAc (5ml) After stirringfor 24 h the solution was concentrated and the residue waswashed with a mixture of diethyl ether and MeOH (20 1)to remove less polar impurities The residue was dried andpurified sequentially byMPLC on reverse phase C-8 silica gel(H2OCH3CN= 1 1 to 1 2) and RP-HPLC (GRACEVYDACC18 20mlminminus1 10 to 60 CH3CN in H2O 220 nm)Thepurified product was lyophilized to give (8) (76mg 58) asa light greenish-yellow sticky solid (HCl salt)

Analytical HPLC (ZORBAX SB-C8) tR = 21min (flowrate 1 cm3minminus1 UV = 220 nm isocratic CH3CN H2O= 30 70) purity 99+ UV (H2O) 120582max 492 nm 120576 =18333 cmminus1Mminus1) 1H-NMR (300MHz MeOD ppm) 120575 089ndash091 (m 6H) 101ndash180 (m 45H) 200ndash209 (m 16H) 238ndash243 (m 16H) 329ndash332 (m 16H partially overlapped withMeOD peak) 348ndash388 (m 6H) 403ndash455 (m 9H) 500ndash541 (m 2H) 666ndash678 (m 6H) 709ndash734 (m 4H) 770 (m1H) 780 (m 1H) 806 (m 1H) MALDI-TOF-MS [M + Na]+calcd for C104H171N31O18SNamz 219830 found 219896

Fluoresceinyl (S)-2-(2-(4-Isobutylphenyl)propanamido)-ace-tate (9) A solution of (S)-(+)-ibuprofen (70mg 034mmol)fluorescein amine (129mg 037mmol) EDC (97mg051mmol) and DMAP (21mg 017mmol) in DMF (2ml)was stirred at rt under N2(g) After 24 h the solution wastreated with EtOAc and washed several times with saturatedaq NaHCO3 water and brine The organic phase was driedand concentrated to give the crude product which was


purified by column chromatography on silica gel elutingwith 1 20MeOHCH2Cl2 to afford (9) (140mg 77) as ayellowish sticky liquid119877119891 054 (CH2Cl2 MeOH = 10 1) 1H-NMR (300MHz

CDCl3 ppm) 120575 090 (d J = 66Hz 6H) 159 (d J = 72Hz3H) 185 (m 1H) 246 (d J = 72Hz 2H) 654ndash672 (m4H) 678ndash695 (m 4H) 711 (m 3H) 728 (m 2H) 13C-NMR(75MHz CDCl3 ppm) 120575 187 227 305 318 369 453 4551032 1087 1103 1103 1108 1130 1172 1172 1177 1227 12491275 1283 1293 1294 1299 1372 1412 1429 1487 15221523 1526 1595 1631 1703 1732MS (FAB) [M+H]+ calcdfor C33H29NO6 mz 53520 found 53519

23 Biological Assays

231 Materials High glucose Dulbeccorsquos modified Eaglersquosmedium (DMEM) Dulbeccorsquos phosphate buffered saline(PBS pH 74) fetal bovine serum (FBS) and trypsinEDTAwere obtained from Invitrogen Milli-Q purified water(182MΩ) was used to prepare all of the solutions

232 Cell Culture HeLa cells were incubated at 37∘C in ahumidified 5 CO2 containing air environment in DMEMand 10 (vv) FBS with penicillin The subculture wasperformed every 2-3 days until the cells were grown tosubconfluence

233 Cellular Uptake Experiment HeLa cells (1 times 105 cellsper well) were seeded into a 35mm cover glass bottomeddish (SPL Ltd Republic of Korea) and incubated for 24 hAfter removing the media HeLa cells were washed with coldPBS (times1) The cells were incubated for 30min at 37∘C in 3mlof DMEM containing 10 120583M of compound (8) or (9) Afterincubation the cell media were removed and HeLa cells werewashed five times with cold PBS replaced with cold PBSand then observed directly on the cover glass bottom dishConfocal laser scanning microscope was performed by usingan Olympus Fluoview FV1000 equipped with an NA 13040x planApo oil immersion lens Fluorescence of FITC wasanalyzed and collected using the excitation bands at 488 nmand emission bands from 500 to 530 nm All the experimentwas performed in triplicate

234 Mouse Tissue Biodistribution Compound (8) or (9)(946mg kgminus1)was dissolved in sterile distilledwater (500120583L)and each solution was injected intraperitoneally (ip) intoeight-week-old mice (C57BL6 22 g) After 20min theadministered mice were perfused with paraformaldehyde(4) in PBS (pH 74) and the major organs (brain heartlung kidney spleen and liver) were incubated overnight in asolution of sucrose (05M) in PBS Placed in cryoprotectantthey were cut into 15 120583m sections with a cryostat and trans-ferred to coated glass slides After drying each section waswashed with PBS treated with Triton X-100 (03) for 15minat rt and then analyzedwith anAxioplan2 fluorescence imag-ing microscope As a control triple distilled water (500120583L)was also injected into the mouse and treated in the sameprocess Green fluorescence from FITC labeled compounds

in each tissue was compared with autofluorescence fromthe control All mouse experiments were performed in thePOSTECH animal facility in compliance with the relevantlaws and institutional guidelines

235 Plasma Stability Test A homogenous solution of (8)in distilled water (500120583gmlminus1) was prepared for the deter-mination of plasma stability Aliquots were withdrawn andadded into plasma (from human Sigma USA) resulting ina final concentration of 50 120583gmlminus1 which was vortexed andincubated at 37∘C for predetermined times (0 3 6 9 12 15 1821 and 24 h) Then aliquots of plasma solution (200120583l) werewithdrawn to be mixed with 1ml MeOH to prevent furtherhydrolysis of (8) To the solution was added biotin (internalstandard) in distilled water (100 120583l 5 120583gmlminus1) followed bycentrifugation at 2000 rpm for 10min The supernatant wascollected and dried under air flow The residue was dissolvedin distilled water (2ml) and centrifuged at 2000 rpm for10min The obtained supernatant was analyzed using HPLCby the following method

236 HPLC Analysis As described earlier a clear super-natant was obtained and 10 120583l was injected into HPLCsystem The UV detector was operated at 260 nm andthe flow rate used was 10mlminminus1 for 10ndash15min runningtime The isocratic mobile phase system was composed ofMeOHH2O (3070) and the column ovenwasmaintained at37∘Cwith an analytical C18-column (Agilent ZORBAX 46times250mm 5 120583m) At each interval HPLC chromatograms wereobtained and the remained percentage of (8) was calculatedby subtracting the released ibuprofen concentration based onarea under curve from the sum of initial concentration of (8)and internal standard

3 Results and Discussion

31 Synthesis of Ibuprofen Conjugate Since the S-(+) enan-tiomer of ibuprofen (dexibuprofen) is more active in inhibit-ing the COX enzymes the S-(+) isomer was used as amodel drug of NSAID Attempting to increase the efficiencyof the coupling of ibuprofen into the sterically crowdedscaffold (sorbitol) we synthesized an ibuprofen derivativeusing glycine as a spacer (Scheme 1) S-(+)-Ibuprofenwas firstcoupled with ethylglycinate to form compound (1) which wasfurther treated with lithium hydroxide to give the compound(2) in accordance with the reported procedure [26] The acid(2) was used as a precursor of ibuprofen conjugate

From our previous study we had observed that thebranched side chains with bis-guanidine moiety at the ter-minal and five-carbon units in the 120596-amino acid chain givebetter efficiency toward cell penetration than other versionsof derivatives [22] Therefore we synthesized the ibuprofenattached sorbitol derivative with five-carbon attached sidechains (Scheme 2) To synthesize compound (3) we startedfrom the commercially available d-glucose Following severalsteps in the reported procedure [22] d-glucose was reducedto acylated sorbitol which was consecutively coupled withCbz-protected aminocaproic acid to form compound (3) To


O

HO

O

HNO

O

O

HNHO

O

(1)(S)-Ibuprofen

(2)

(b)

(a)

Scheme 1 (a) Glycine ethylestersdotHCl EDC DMAP DMF and Et3N rt 24 h 99 (b) LiOH and THF-H2O (5 1) rt 36 h 60

remove the trityl group selectively in (3) in the presenceof eight Boc groups gradients of CH2Cl2MeOH mixturewere eluted through a flash SiO2 column packed in hexanecontaining 1 TFA at the top and hexane containing 1 Et3Non the bottom [27] As a result compound (4)was obtained in73 yield after column chromatographyThe hydroxyl groupof (4) was coupled with the ibuprofen derivative (2) in thepresence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and 4-dimethylaminopyridine (DMAP) in DMF toafford the conjugate (5) in moderate yield The conjugationwas covalently formed via an ester bond which can be easilyhydrolyzed to release ibuprofen after reaching the targetDeprotection of N-Cbz group in the compound (5) wasperformed using 10PdC and hydrogen gas under 50 psito give the amino compound (6) which did not requirefurther purification In order to attach a fluorescent probe thecompound (6) was treated with fluorescein-5-isothiocyanate(FITC) and Et3N in a mixed solvent THFethanol to givethe compound (7) in moderate yield Finally the compound(7) was treated with ethylacetate saturated with HCl (gas)to remove all the Boc groups of the guanidine moieties toyield compound (8) as hydrochloride salts The resulting salt(8) was purified by medium pressure liquid chromatography(MPLC) using a reverse-phase (C8) column In order to com-pare the delivery efficiency between the compound (8) andthe blank compound (ie without sorbitol-based moleculartransporter) we had synthesized the compound (9) by EDCcoupling strategy at room temperature (Scheme 3) Then (9)was further purified by column chromatography on silica gel

32 Cellular Uptake Study of the Ibuprofen Conjugate Inorder to check whether the synthetic conjugate (8) can crossthe cell membrane and translocate itself inside cells weinvestigated its intracellular delivery properties by means ofconfocal microscope (Olympus Fluoview FV1000 NA 13040x) The conjugate (8) (10 120583M in PBS) was added to themedia of live HeLa cells and was incubated together at 37∘Cfor 30min Then the cells were washed with cold PBS twiceand the cellular internalization of the compound and itslocalization could be examined by the fluorescence of thelabeled fluorescein Figure 2 shows strong green fluorescenceappearing mainly in the cytoplasm and this indicates (8)

can cross the cellular membrane and diffuse efficiently in thecytoplasm However unlike the ibuprofen conjugate whereibuprofen conjugated only with FITC (9) was incubated inHeLa cells in the samemanner no fluorescence was observedinside the cells This result confirms the guanidine moietiesin the conjugate structure are completely essential in deliv-ering ibuprofen into cells Although the exact mechanism ofcellular uptake mediated by guanidine is very complicatedand sometimes remains controversial according to recentpublications it is assumed that at the initial stage of celluptake a strong association takes place between the cationicguanidine and anionic cell membrane constituents such assulfate carboxylate and phosphate groups [28 29]

33 Mouse Tissue Distribution Experiment The possibility ofBBB penetration of the conjugate (8) was tested by studyingthe tissue distribution in mice The conjugate (8) (HCl salt944mg kgminus1) was dissolved in sterile distilled water andthe solution was injected intraperitoneally into an 8-week-old mouse (C57BL6) After 20min the injected mouse wastranscardially perfused with paraformaldehyde in phosphatebuffered saline solution (pH 74) The major organs such asbrain heart lung kidney spleen and liver were incubatedovernight in a solution of sucrose (05M) in PBS Placedin cryoprotectant the tissues were cut into 15 120583m sectionswith a cryostat and transferred to coated glass slides Afterdrying sections were treated with Triton X-100 for 15minand then analyzed with fluorescence microscope (Figure 3)As a control study only PBS was injected to another mouseIn the previous study the sorbitol G8 transporter showedpreferred distribution patterns to heart and brain than toother tissues [22] Conjugate (8) shows preferred locations tobe in the brain and the kidney while much less distributionswere observed in the spleen and the liver and none in theheart and the lungThis shows the conjugate can readily crosstheBBB andpenetrate into themouse brain Similarly (9)wasused for the experiment with the same procedure but showedno distribution in the brain We presume that the guanidine-rich region of (8) forms noncovalent bonds with brain cellsurface units through charge pairing and hydrogen bondingAfter this interaction (8) might cross the BBB by endocytosis[28 30]


OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2

Scheme 2 (a) SiO2 with hexane-1 TFA and hexane-1 Et3N 73 (b) (2) EDC DMAP and DMF rt 48 h 52 (c) 10 PdC H2(g) (50 psi)and MeOH-CH2Cl2(9 1) rt 12 h 88 (d) FITC-I Et3N THF and EtOH rt 36 h 60 (e) HCl(g) in EtOAc rt 24 h 58

O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)

Scheme 3 (a) Aminofluorescein EDC DMAP and DMF rt 24 h 77


(a) (b) (c)

(d) (e)

Figure 2 Cellular uptake of (8) and (9) in live HeLa cells (a) fluorescence image (b) DIC (differential interference contrast) image (c) mergeimage of (a) and (b) from (8) (ibuprofen conjugate) treated cells (d) fluorescence image and (e) DIC image from (9) (control) treated cellsScale bar 20 120583m

34 In Vitro Plasma Stability Test To determine the stabilityof the ibuprofen conjugate (8) was incubated in the presenceof human plasma Initially ibuprofen biotin as internalstandard blank plasma and (8) were analyzed respectivelyby HPLC at 260 nm to determine each retention time (min)ibuprofen 39 biotin 31 blank plasma 40 and 52 and (8)78

Compound (8) was dissolved in distilled water (500120583gmlminus1) and then aliquots were mixed with plasma to bea concentration of 20120583gmlminus1 During incubating solutionat 37∘C aliquots (200 120583l) were withdrawn periodically andmixed with MeOH (1ml) and biotin (100 120583l 5 120583gmlminus1) as aninternal standard After centrifugation the supernatant wascollected and 10 120583l of the solution was injected for RP-HPLCequipped with a C18-analytical column Samples detecting at260 nm showed a slight shift of retention time of each speciesand particularly the released ibuprofen was identified by thepeak with retention time at 396min (Figure 4(a)) After 3 hincubation compound (8) underwent hydrolysis around 5with respect to the initial time (0 h) With the increase oftime however the remaining percentage decreased slowlysim8 in next 21 h incubation time (Figure 4(b))This indicates(8) has sufficient stability and long half life time in plasma

and the compound could gain higher possibility to reach theBBB

4 Conclusions

In conclusion ibuprofen conjugate (8) was prepared usinga sorbitol-based G8 molecular carrier and showed gooddistribution toward mouse brain Although there is nocure or protecting agent for neurodegenerative disease yetNSAIDs are being reported to give beneficial neuroprotectioneffects To deliver a drug into brain in optimal dose and tominimize side effects an efficient and specific delivery systemovercoming the BBB is highly required We have succeededin delivering ibuprofen conjugate to mouse brain and ourprodrug methodology is applicable to virtually all otherNSAIDs such as flurbiprofen Further studies are in progressto demonstrate the delivery of other popular NSAIDs to thebrain and the central nervous system

Competing Interests

The authors declare that they have no competing interests


Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)

Figure 3 Distribution tests in mouse tissues (left column control middle (8) and right (9)) Fluorescence micrographs of ((a)ndash(c)) brain((d)ndash(f)) heart ((g)ndash(i)) lung ((j)ndash(l)) kidney ((m)ndash(o)) spleen and ((p)ndash(r)) liver tissue sections isolated frommice 20min after ip injectionExposure times (ms) brain 5000 heart 1000 lung 5000 kidney 2000 spleen 2000 and liver 2000 120582max = 488 nm (green fluorescencefrom FITC)

Acknowledgments

The authors would like to thank Dr Sung-Kee Chung (hon-orary professor at Pohang University of Science and Tech-nology) for his kind advice and interest This research was

supported by Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded bythe Ministry of Science ICT amp Future Planning (NRF-2015R1C1A1A01054657) and partly supported by the Indus-try Technology RampD Program of MOTIEKEIT [10051080


50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)

Figure 4 (a)HPLC chromatogramof biotin (internal standard) and(8) in plasma after 3 h incubation time retention time of biotin andplasma 338min ibuprofen 396min and (8) 640min and (b) theremained percentage of (8) in plasma over incubation time

development of mechanical UI device core technology forsmall and medium-sized flexible display] This work was alsosupported by the SoonchunhyangUniversity Research Fund

References

[1] J A Mitchell P Akarasereenont CThiemermann R J Flowerand J R Vane ldquoSelectivity of nonsteroidal antiinflammatorydrugs as inhibitors of constitutive and inducible cyclooxyge-naserdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 24 pp 11693ndash11697 1993

[2] R J Flower ldquoThe development of COX2 inhibitorsrdquo NatureReviews Drug Discovery vol 2 no 3 pp 179ndash191 2003

[3] A H Moore M J Bigbee G E Boynton et al ldquoNon-steroidalanti-inflammatory drugs in Alzheimerrsquos disease and Parkinsonrsquosdisease reconsidering the role of neuroinflammationrdquo Pharma-ceuticals vol 3 no 6 pp 1812ndash1841 2010

[4] J L Eriksen S A Sagi T E Smith et al ldquoNSAIDs andenantiomers of flurbiprofen target 120574-secretase and lower A12057342in vivordquo The Journal of Clinical Investigation vol 112 no 3 pp440ndash449 2003

[5] A Lleo O Berezovska L Herl et al ldquoNonsteroidal anti-inflammatory drugs lower A12057342 and change presenilin 1 con-formationrdquoNatureMedicine vol 10 no 10 pp 1065ndash1066 2004

[6] MH DeLegge and A Smoke ldquoNeurodegeneration and inflam-mationrdquo Nutrition in Clinical Practice vol 23 no 1 pp 35ndash412008

[7] H Chen S M Zhang M A Hernan et al ldquoNonsteroidal anti-inflammatory drugs and the risk of Parkinson diseaserdquoArchivesof Neurology vol 60 no 8 pp 1059ndash1064 2003

[8] F L Heppner R M Ransohoff and B Becher ldquoImmune attackthe role of inflammation in Alzheimer diseaserdquo Nature ReviewsNeuroscience vol 16 no 6 pp 358ndash372 2015

[9] T Wyss-Coray and L Mucke ldquoIbuprofen inflammation andAlzheimer diseaserdquo Nature Medicine vol 6 no 9 pp 973ndash9742000

[10] E E Tuppo and H R Arias ldquoThe role of inflammation inAlzheimerrsquos diseaserdquo The International Journal of Biochemistryamp Cell Biology vol 37 no 2 pp 289ndash305 2005

[11] G P Lim F Yang T Chu et al ldquoIbuprofen suppresses plaquepathology and inflammation in a mouse model for Alzheimerrsquosdiseaserdquo The Journal of Neuroscience vol 20 no 15 pp 5709ndash5714 2000

[12] S C Vlad D R Miller N W Kowall and D T FelsonldquoProtective effects of NSAIDs on the development of Alzheimerdiseaserdquo Neurology vol 70 no 19 pp 1672ndash1677 2008

[13] X Gao H Chen M A Schwarzschild and A Ascherio ldquoUseof ibuprofen and risk of Parkinson diseaserdquo Neurology vol 76no 10 pp 863ndash869 2011

[14] Z Xuan L Xing G Tao S Xun and Z-R Zhang ldquoIn vitroand in vivo investigation of dexibuprofen derivatives for CNSdeliveryrdquo Acta Pharmacologica Sinica vol 33 no 2 pp 279ndash288 2012

[15] Y Deguchi H Hayashi S Fujii et al ldquoImproved brain deliveryof a nonsteroidal anti-inflammatory drug with a syntheticglyceride ester a preliminary attempt at a CNS drug deliverysystem for the therapy of Alzheimerrsquos diseaserdquo Journal of DrugTargeting vol 8 no 6 pp 371ndash381 2000

[16] B P Imbimbo ldquoWhy did tarenflurbil fail in Alzheimerrsquos dis-easerdquo Journal of Alzheimerrsquos Disease vol 17 no 4 pp 757ndash7602009

[17] D Zheng X Shuai Y Li et al ldquoNovel flurbiprofen derivativeswith improved brain delivery synthesis in vitro and in vivoevaluationsrdquo Drug Delivery vol 23 no 7 pp 2183ndash2192 2014

[18] JMR ParepallyHMandula andQR Smith ldquoBrain uptake ofnonsteroidal anti-inflammatory drugs ibuprofen flurbiprofenand indomethacinrdquo Pharmaceutical Research vol 23 no 5 pp873ndash881 2006

[19] S Lehrer ldquoNasal NSAIDs for Alzheimerrsquos diseaserdquo AmericanJournal of Alzheimerrsquos Disease amp Other Dementias vol 29 no5 pp 401ndash403 2014

[20] S C Ghosh B Kim J Im et al ldquoMitochondrial affinityof guanidine-rich molecular transporters built on m120574o- andsc120574llo-inositol scaffolds stereochemistry dependencyrdquo Bulletinof the Korean Chemical Society vol 31 no 12 pp 3623ndash36312010

[21] J Im S Kim Y-H Jeong et al ldquoPreparation and evaluationof BBB-permeable trehalose derivatives as potential therapeuticagents for Huntingtonrsquos diseaserdquoMedicinal Chemistry Commu-nications vol 4 no 2 pp 310ndash316 2013

[22] K K Maiti W S Lee T Takeuchi et al ldquoGuanidine-containingmolecular transporters sorbitol-based transporters show highintracellular selectivity toward mitochondriardquo AngewandteChemiemdashInternational Edition vol 46 no 31 pp 5880ndash58842007


[23] J Im K KMaitiW Kim K-T Kim and S-K Chung ldquoCellularuptake properties of the complex derived from quantum dotsand G8 molecular transporterrdquo Bulletin of the Korean ChemicalSociety vol 32 no 4 pp 1282ndash1292 2011

[24] J Im W Kim K-T Kim and S-K Chung ldquoPreparation of a31015840-azido-31015840-deoxythymidine (AZT) derivative which is bloodndashbrain barrier permeablerdquo Chemical Communications no 31 pp4669ndash4671 2009

[25] J Im G BiswasW Kim K-T Kim and S-K Chung ldquoA blood-brain barrier permeable derivative of 5-fluorouracil prepara-tion intracellular localization and mouse tissue distributionrdquoBulletin of the Korean Chemical Society vol 32 no 3 pp 873ndash879 2011

[26] S Bhuniya Y J Seo and B H Kim ldquo(S)-(+)-Ibuprofen-basedhydrogelators an approach toward anti-inflammatory drugdeliveryrdquo Tetrahedron Letters vol 47 no 40 pp 7153ndash71562006

[27] A K Pathak V Pathak L E Seitz K N Tiwari M S Akhtarand R C Reynolds ldquoA facile method for deprotection of tritylethers using column chromatographyrdquo Tetrahedron Letters vol42 no 44 pp 7755ndash7757 2001

[28] P A Wender W C Galliher E A Goun L R Jones and T HPillow ldquoThe design of guanidinium-rich transporters and theirinternalization mechanismsrdquo Advanced Drug Delivery Reviewsvol 60 no 4-5 pp 452ndash472 2008

[29] J B Rothbard T C Jessop and P A Wender ldquoAdaptivetranslocation the role of hydrogen bonding and membranepotential in the uptake of guanidinium-rich transporters intocellsrdquo Advanced Drug Delivery Reviews vol 57 no 4 pp 495ndash504 2005

[30] E Wexselblatt J D Esko and Y Tor ldquoOn guanidinium andcellular uptakerdquo The Journal of Organic Chemistry vol 79 no15 pp 6766ndash6774 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


BBB BBB BBB

Blood

Brain

Ibuprofen

Ibuprofen-HOO

conjugate

Figure 1 The presence of BBB prevents the ibuprofen internaliza-tion into the brain Our objective is to make ibuprofen conjugatepass through the BBB

clinical research data from almost 250000 cases showedthose who took NSAID for more than five years were morethan 40 less likely to develop Alzheimerrsquos [12] The studyalso revealed that among the NSAIDs taken by patientsibuprofen showed the most profound effect Furthermore astudy suggests taking ibuprofen regularly to be also associatedwith lowering the risk of the development of Parkinsonrsquosdisease [13] The study reported that those taking ibuprofenregularly more than six years showed a 38 lower risk ofdeveloping the disorder than nonusers and other NSAIDssuch as aspirin and paracetamol did not show any effect tothe patients

However there are many challenges to be overcome foribuprofen and other NSAIDs to be used as treatments forneural disorders First the presence of blood-brain barrier(BBB) has been reported to significantly limit the brainpenetration of NSAIDs (Figure 1) [14ndash17] The BBB betweencerebral blood and the interstitial fluid of the brain actsas protection from an influx of pathogens as well as smalluseful molecular drugs like NSAIDs In addition ibuprofenis ionized at physiological pH and circulates as anions whichare extensively bound to plasma proteins by noncovalentassociations [18 19] As a consequence brain penetration ofibuprofen was found to be low in rats showing the total brainto plasma ratio being less than 002 Therefore ibuprofenreaching the human brain exists in only a tiny amount andhigh doses might be required to attain a certain level ofdrug efficacy toward neural disorders Also long term useof ibuprofen can cause a number of side-effects such ascardiovascular and gastrointestinal risks

Therefore to make ibuprofen a more potent drug toprevent or alleviate neurodegenerative diseases even in lowdoses delivery of ibuprofen to brain should be effective andibuprofen must penetrate the BBB and gain access into thepatientrsquos brain

We have recently developed several types of guanidine-richmolecular carrierswith highmolecularweights and goodwater solubility that readily cross the BBB anddisplay efficientdistributions in themouse brain [20 21] In particular the G8(having eight guanidine groups) molecular carrier based on

carbohydrate such as sorbitol was found to be very effectivein delivering conjugated drugs to mouse brain [22ndash25] Inthe present study employing the same molecular carrierwe prepared the ibuprofen conjugate and studied its BBBpermeability by mouse tissue distribution study

2 Experimental

21 Materials and Methods in Synthesis Analytical TLCwas performed on Merck 60 F254 silica gel plate (025mmthickness) and visualized under UV light andor by sprayingwith a 5 solution of phosphomolybdic acid (PMA) inethanol or ninhydrin specific for amino compounds followedby charring with a heat gun Column chromatography wasperformed on Merck 60 silica gel (70ndash230 or 230ndash400mesh)and MPLC on Fluka 100 C8-reversed phase silica gel NMRspectra were recorded on a Bruker ASPECT 300 instrumentoperating at 300MHz for 1H 75MHz for 13C Chemicalshifts were reported in parts per million (ppm) relative totetramethylsilane (TMS) or deuterium oxide (D2O) whichwere used as internal and external standard for the 1HNMRMass spectra were recorded on fast atom bombardment(FAB) conditions In case of highmass spectral dataMALDI-TOF were obtained on a Micromass MDI at the Biomolec-ular Diversity Core Facility (POSTECH) Analytical HPLCwas performed on Agilent 1100-HPLC Chemstation with ananalytical column ZORBAX C8-monomeric (BU-300 5 120583m300 A 46 times 250mm)

22 Characterization of Synthetic Compounds(S)-Ethyl-2-(2-(4-isobutylphenyl)propanamido)acetate (1) Asolution of (S)-(+)-ibuprofen (140mg 068mmol) glycineethylester hydrochloride (104mg 075mmol) EDC (195mg102mmol) triethylamine (104ml 0748mmol) and DMAP(42mg 034mmol) in DMF (2ml) was stirred at rt underN2(g) After stirring for 24 h the solution was treated withethylacetate and washed several times with saturated aqNaHCO3 water and brine The organic phase was driedover Na2SO4 and concentrated to give the crude productwhich was purified by column chromatography on silica geleluting with 1 5 EtOAchexane to afford (1) (199mg 99) asa colorless liquid119877119891 054 (Hexane EtOAc = 7 3) 1H-NMR (300MHz

CDCl3 ppm) 120575 099 (d J = 66Hz 6H) 123 (t J = 69Hz3H) 151 (d J = 72Hz 3H) 184 (m 1H) 244 (d J = 72Hz2H) 360 (q J = 72Hz 1H) 394 (dd J = 54Hz J = 126Hz2H) 415 (q J = 69Hz 2H) 711 (d J = 81Hz 2H) 722 (d J= 81Hz 2H) 13C-NMR (75MHz CDCl3 ppm) 120575 139 183222 300 413 449 462 611 1272 1294 1381 1405 16971745 MS (FAB) [M + H]+ calcd for C17H25NO3 mz 29218found 29220

(S)-2-(2-(4-Isobutylphenyl)propanamido)acetic acid (2)Com-pound (1) (195mg 067mmol) and lithiumhydroxide (16mg067mmol) were added to 6ml solution of THF H2O (5 1)and stirred at rt for 36 h Then the reaction mixture wasevaporated in vacuo extracted with EtOAc and washedwith saturated 1N HCl water and brine The organic phasewas dried over Na2SO4 and concentrated to give the crude































O

HO

O

HNO

O

O

HNHO

O

(1)(S)-Ibuprofen

(2)

(b)

(a)







OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2


O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)



(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of































O

HO

O

HNO

O

O

HNHO

O

(1)(S)-Ibuprofen

(2)

(b)

(a)







OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2


O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)



(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















O

HO

O

HNO

O

O

HNHO

O

(1)(S)-Ibuprofen

(2)

(b)

(a)







OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2


O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)



(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


O

HO

O

HNO

O

O

HNHO

O

(1)(S)-Ibuprofen

(2)

(b)

(a)







OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2


O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)



(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


OHOHO

OH

OHOH

d-Glucose

O

OR

OROROR

OR

OR

RO RO

OTr

ONHCbz

RO

RO

RO

NHCbz

NHCbz5

(3)

(a)

O

OH

O

5

(4)

(b)

O

O

5

(5)

(c)

O

O

5

(6)

(d)

(e)

O

O

NH-FITC5

(7)

O

ONH-FITC5

(8)

N

NH

HN

NHBoc

NHBoc

NBoc

NBoc

O

5R =

N

NH

HN

O

5

O

HN

O

OR

OROR

OR

OROR

OR

OROR

OR1

OR1OR1

OR1

OR2

OR2

OR2

R1 =

R2 =

NH2

NH2

NH ∙ HCl

NH ∙ HCl

R2O

NH2


O

HO

(S)-Ibuprofen

(a)O

HO

HO

OO

O

HN

(9)



(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b) (c)

(d) (e)





4 Conclusions


Competing Interests



Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Brain

Heart

Lung

Kidney

Spleen

Liver

Control (8) (9)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)


Acknowledgments




50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


50

0

(mV

)

(min)

338

396

640

15012510075502500

(a)

3 6 9 12 15 18 21 240t (hr)

80

82

84

86

88

90

92

94

96

98

100

Rem

aine

d pe

rcen

tage

()

(b)



References









































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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Synthesis of Ibuprofen Conjugated Molecular Transporter ...downloads.hindawi.com/journals/jchem/2017/4746158.pdf · ResearchArticle Synthesis of Ibuprofen Conjugated Molecular Transporter