Aromatic radiofluorination and biological evaluation of 2-aryl-6-[ 18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

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Aromatic radiofluorination and biological evaluation of 2-aryl-6-[ 18F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for β-amyloid plaques

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   Aromatic radiofluorination and biological evaluation of 2-aryl-6-[ 18 F]fluorobenzothiazoles as a potential positron emission tomography imaging probe for   b -amyloid plaques Byung Chul Lee a,b , Ji Sun Kim a , Bom Sahn Kim c , Ji Yeon Son a , Soo Kyung Hong a , Hyun Soo Park a ,Byung Seok Moon a,b , Jae Ho Jung a , Jae Min Jeong b , Sang Eun Kim a,b, ⇑ a Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea b Institute of Radiation Medicine, Seoul National University Medical Research Center, Seoul National University, Seoul 110-799, Republic of Korea c Department of Nuclear Medicine, Ewha Womans University College of Medicine, Seoul 158-710, Republic of Korea a r t i c l e i n f o  Article history: Received 20 January 2011Revised 11 March 2011Accepted 12 March 2011Available online 21 March 2011 Keywords: b -AmyloidAlzheimer’s diseaseDiaryliodonium saltPET a b s t r a c t To develop agents for radionuclide imaging A b  plaques in vivo, we prepared three fluorine-substitutedanalogs of arylbenzothiazole class; compound  2  has a high affinity for A b  ( K  i  =5.5nM) and the specificbinding to A b  in fluorescent staining. In preparation for the synthesis of these arylbenzothiazole analogsin radiolabeled form as an A b  plaques-specific positron emission tomography (PET) imaging probe, weinvestigated synthetic route suitable for its labeling with the short-lived PET radionuclide fluorine-18( t  1/2  =110min) and diaryliodonium tosylate precursors ( 12 ,  13a – e  and  14 ). 2-Aryl-6-[ 18 F]fluorobenzo-thiazoles ([ 18 F] 1 – 3 ) were synthesized in efficiently short reaction times (40–60min) with high radio-chemical yields (19–40%), purities (>95%) and specific activities (85–118GBq/ l mol). Tissue distributionstudies showed that high radioactivity of [ 18 F] 2  accumulated in the brain with rapid clearance in healthymice. Radioactivemetaboliteswereanalyzedinbrainsamplesof miceandcorrespondedto81%ofparentremained by 30min after a tail-vein injection. These results suggest that [ 18 F] 2  is a promising probe forevaluation of A b  plaques imaging in brain using PET.   2011 Elsevier Ltd. All rights reserved. 1. Introduction AD is a progressive and fatal neurodegenerative disease thatleads to brain disorder; half of the affected patients suffer fromdementia, cognitive impairment, and memory loss. The most seri-ous risk factor of AD is that it becomes more pronounced withincreasing age of the patient, and this is a concern because of theincreasing life expectancy of populations worldwide; conse-quently, there is an increasingly high incidence of AD in the olderpopulation. Approximately 5% of those over 65years are affected,whileover 20–30%of thoseover 80years showsignsof dementia. 1 It has been reported that the main event in the pathogenesis of ADis the formation and accumulation of aggregates of A b  peptides inthe brain. 2,3 Therefore, A b  plaque, which seems to play an impor-tant neuropathological role in AD, is mainly comprises an aggrega-tion of the A b  peptide. 4 An A b  plaques-specific imaging probewould be useful for the diagnosis of AD at an early to moderatestage, as well as the diagnosis of MCI and the monitoring of thetherapeutic efficacy AD treatment. There has been an ongoingworldwide search for a clinically useful radiolabeled A b  plaqueprobe in order to develop noninvasive in vivo PET imaging forAD. To this end, many specific ligands have been developed andevaluated for the imaging of A b  plaques. One of the lead structuralmotifs, 2-(4 0 -([ 11 C]methylaminophenyl)-6-hydroxybenzothiazole(known as [ 11 C]PIB, Fig. 1) has already shown promising resultsin clinical trial. 5–7 Despite the promising clinical results in ADpatients, PIB remains suboptimal on account of its labeling withthe short-lived carbon-11 (half life=20min), which limits itsavailability to centers equipped withanon-site cyclotron. To over-comethislimitationof[ 11 C]PIB,duetotheshorthalflifeofcarbon-11, many research groups tried to introduce fluorine-18 (half life=110min) into their target compound. Thus far, the com-pounds reported to have a high affinity for A b  plaque and whichcontained fluorine-18 were fluorine-18 labeled BTA analogs(GE-067 and 6-OH-4 0 -FP-BTA), 8–10 FDDNP, BAY94–9174, andAV-45 (Fig. 1). 11–13 Among these radioligands, FDDNP was known to bind to bothA b  plaques at different binding sites and in intracellular neurofi-brillary tangles; it does not selectively measure a specificpathologic component of AD patients. 11 The recently reportedfluoropegylated stilbene derivatives ([ 18 F]BAY94-9172 andAV-45) are currently under a phase II and phase III, respectively.The most commonly studied and most promising of the reported 0968-0896/$ - see front matter   2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.03.029 ⇑ Corresponding author. Tel.: +82 31 787 7671; fax: +82 31 787 4018. E-mail address:  kse@snu.ac.kr (S.E. Kim).Bioorganic & Medicinal Chemistry 19 (2011) 2980–2990 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc  compounds, members of the novel 2-aryl-6-hydroxybenzothiazoleclass ([ 18 F]GE-067, including PIB), showed a high initial brain up-take, and a particularly high binding affinity to A b  plaque, andare currently being used in clinical trials (phase III).Tracing the history of the studies for the fluorine-18 labeledcompounds as an A b  plaque imaging probe, it is believed that2-(4-aminophenyl)-benzothiazole (BTA) may have potential as aparentalcompoundandaromaticfluorine-18labelingintotheben-zothiazole moiety or aniline in BTA is essential for in vivo stabilityand retaining its biological activities as PET imaging probe for A b plaque.In order to achieve this, preliminary reports on fluorine-18introduced at the  ortho - or  para -position in the right aromatic ringof BTA analogs ([ 18 F]GE-067 and 6-hydroxy-2-(4 0 -[ 18 F]fluoro-phenyl)-benzothiazoles) were developed. 10 In addition to, a previ-ous article by Henriksen, et al., described the preparation of a 11 C-labeled 5-fluorine substituted benzothiazole analog as animaging probe and mentioned the fluorine at 5-position metaboli-cally stabilized BTA analog compare to [ 11 C]PIB is known to berapidlymetabolizedtotheC6-sulfonatedderivatives. 14,15 Recently,Zheng et al. also described the preparation of 6-fluroine substi-tuted benzothiazole analog as an imaging probe and showed theircompounds have higher binding affinities to A b  aggregates thanPIB. 16 It was interesting to note that fluorine effectively replacedhydroxyl group while continuing to manifest comparable activi-ties, albeit with different properties, in numerous examples. 17,18 Although6-fluroinesubstitutedbenzothiazoleanalogshowedhighbindingaffinitytoA b  plaque,theyfailedfluorine-18labelinginthebenzothiazolemoietyoftheircompoundsbecausetheintroductionof fluorine-18 into benzothiazole ring was impossible by generalaromatic fluorination.Gratifying, diaryliodoniumsalt precursors canbe useful to labelfluorine-18 at aromatic position in aryl compounds, which haveproven difficult to label using the general aromatic fluorine-18labeling methods in the previous works of our group 19,20 and thePike group. 21 Therefore, we designed three 6-[ 18 F]fluorine labeled BTA ana-logs instead of the 6-hydroxy group in [ 11 C]PIB and synthesizedvarious diaryliodonium tosylate precursors to allowaromatic fluo-rine-18 labeling into the benzothiazole ring. In this investigation,we prepared compounds  1–3  and [ 18 F] 1–3  using two approaches:the first (Scheme 2) involved the preparation of authentic samplesofthese6-fluoro-substitutedBTAanalogs;thesecondwasthesyn-thesis of precursors (diaryliodonium tosylate) and the radio-syn-thetic procedure for BTA analogs in which fluorine-18 could beintroduced to the precursor at a very late step. Reported hereinare in vitro and in vivo evaluations of novel three 6-[ 18 F]fluoro la-beledBTAanalogs for use as a prospectivePET probe for A b  plaqueimaging in the brain. 2. Results and discussion2.1. Chemical synthesis We considered a number of approaches that would be suitableforthepreparationof6-fluoro-substitutedBTAanalogsinfluorine-18labeledformthat wouldhave the high specificactivityrequiredfor a PET imaging probe. In our system, it was not clear whetherthe benzothiazole substituent at the 6-position would provide SNHONR 1 H PIB :R 1  = CH 3 , R 2  = H GE-067 :R 1  = CH 3 , R 2  = FR 2 CH 3 NCCNNFCH 3 FDDNP BAY94-9174 : R = CH AV-45 : R = NRNOF3HH 3 CSNHO 6-OH-4'-FP-BTA F Figure 1.  Chemical structures of previously reported A b  plaque PET imagingprobes.   +SNIRSNFRXSHNH 2 OHCR 6 : R = NO 2 7 : R = N(CH 3 ) 2 8 : R = NO 2 , 57% 1 : R = NH 2 , 74% 2 : R = NHCH 3 , 35% 3 : R = N(CH 3 ) 2 , 85%(i)(ii)(iii) 9a : R = NO 2 , 95% 9b : R = NCH 3 Boc, 49% 9c : R = N(CH 3 ) 2 , 21%(iv)X = FX =Br 11a : R = NO 2 , 52% 11b : R = NCH 3 Boc, 48% 11c : R = N(CH 3 ) 2 , 65%(v)(vi)  12 : R = NO 2 , 38% 13a : R = NCH 3 Boc, 39%  14 : R = N(CH 3 ) 2 , 13%(ii), 10 SNBrRSNBu 3 SnR-OTs 4 : X = F 5 : X = Br Scheme 1.  Reagents and conditions: (i) DMSO, 180  C, 30min; (ii) SnCl 2 , EtOH, 90  C, 2h; (iii) CH 3 I, K 2 CO 3 , DMSO, 100  C, 16h; (iv) (a) formaldehyde, MeOH, reflux, 2h; (b)NaCNBH 3 , AcOH, room temperature, 1.5h; (c) Boc 2 O, THF, 85  C, 12h; (v) Sn 2 Bu 6 , Pd(0), THF, 85  C, 8h; (iv) Koser’s reagent ( 15a ), CH 3 CN, room temperature, 12h. B. C. Lee et al./Bioorg. Med. Chem. 19 (2011) 2980–2990  2981  sufficient activation for fluoride ion substitution with generalnucleophilic aromatic substitution of a suitable leaving group. 29,30 Wewereunabletoeffectfluorideionsubstitutionatthe6-positionwith a nitro group. Curiously, we were also unable to prepare themore reactive  p -trimethylammonium precursor because of theside compound ( N  -methylbenzothiazole) in the final methylationstep from the dimethylaniline precursor. The limitation of generalaromatic fluorine-18 labeling in our target compounds led us topropose an alternative approach to aromatic fluorination. It isknown that diaryliodonium salts can be used as precursors for flu-oroarenes, 31,32 and their value corresponds well with our reportedfluorine-18 labeled PPARgamma ligand by iodoniumsalts in a pre-vious work. 20 The three 6-fluoro-substituted BTA analogs  1 – 3  were simplysynthesized from 2-amino-5-fluorobenzenethiol  4  using a littlemodification of known methods (Scheme 1). 23,33 6-Fluoro-substi-tuted benzothiazol rings,  8  and  3  were synthesized directly with2-amino-5-fluorobenzenethiol  4  and 4-nitrobenzaldehyde  6  or4-(dimethylamino)-benzaldehyde  7 , respectively, followed byreduction of the nitro compound  8  with tin chloride to producethe corresponding amine  1 . This amine compound proceededN-monomethylation with iodomethane to produce the corre-sponding monomethyl amine  2 . A different approach was neededfor the synthesis of target compounds  1 – 3  in radiolabeled form.The tributyltin analogs  11a – c  can be readily prepared from thebromo compounds  9a – c  in the presence of bis(tributyl)tin and acatalytic quantity of tetrakis(triphenylphosphine)palladium(0). Ata late stage in the synthesis, the fluorine-18 labeling step can rap-idly and efficiently introduce fluorine-18 into appropriate precur-sors that are available in radiolabeled form at high specificactivity because the fluorine-18 ion has a relatively short life (half life=110min). Therefore, we designed a different radiolabelingpathway in which one is a single step with direct aromatic fluo-rine-18 labeling in the synthesis of [ 18 F] 3  and the other is a one-pot two step with aromatic fluorine-18 labeling and reduction ordeprotection in the syntheses of [ 18 F] 1  and [ 18 F] 2 . Therefore, thetributyltin analogs were prepared in different functional groups:dimethylamine 14 ,nitro 12 andBoc-protectedmonomethylamine 13a – e  on the right aromatic ring.To prepare iodonium tosylate precursors suitable for the intro-duction of fluorine-18 ion into the BTA analogs, we reacted the tri-butyltin compounds  11a – c  with a commercially available Koser’sreagent  15a  and various hydroxy(tosyloxy)iodoarenes  15b – e .Theelectron-richhydroxy(tosyloxy)iodoarenesareknowntobeunstable and to decompose violently at room temperature. Thus,we prepared  15b – e  according to the literature and use it immedi-ately to prepare the diaryliodnium tosylate precursors  13b – e under nitrogen atmosphere. Generally, the fluoride anion is SNBu 3 SnNCH 3 Boc 11b +SNINCH 3 BocAr-OTs(i)IOHArOTs 15a : Ar =phenyl 15b : Ar =  p  -anisole 15c : Ar =  p  -toluene 15d : Ar =2-thiophenyl 15e : Ar =3-thiophenyl 13b : Ar = p  -anisole, 56% 13c : Ar = p  -toluene, 82% 13d : Ar = 2-thiophenyl, 57% 13e : Ar = 3-thiophenyl, 70% Scheme 2.  Reagents and conditions: (i)  15b – e , CH 2 Cl 2 , CH 3 CN, room temperature, 12h.  Table 1 Reaction of aromatic radiofluorination with  12 ,  13a , and  14 Entry Precursor a [ 18 F]F  X + Solvent c M  W  or temp Time Yield e 1  14 b [ 18 F]F  Cs + DMF 130  C 10min NP g 2  14  [ 18 F]F  Cs + MeCN 130  C 10min 2.4%3  14  [ 18 F]F  Cs + MeCN 100W 3min 26.7%4  14  [ 18 F]F  K + Kryptofix MeCN 130  C 10min 5%5  14  [ 18 F]F  K + Kryptofix DMF 100W 3min NP g 6  14  [ 18 F]TBAF MeCN d 100W 3min 18.7% f  7  14  [ 18 F]TBAF MeCN 100W 3min 29% f  8  14  [ 18 F]TBAF MeCN 100W 6min 35.6±3.5% ( n  =2) f  9  12  [ 18 F]F  K + Kryptofix MeCN 100W 3min 5%10  12  [ 18 F]TBAF MeCN 100W 6min 35.3±5.7% ( n  =4) f  11  13a  [ 18 F]TBAF MeCN 130  C 10min 9%12  13a  [ 18 F]TBAF MeCN d 130  C 10min 19.3±7.7% ( n  =5) f  13  13a  [ 18 F]F  K + Kryptofix MeCN d 130  C 10min 6.4%14  13a  [ 18 F]F  K + Kryptofix MeCN d 130  C 15min 13.3%15  13a  [ 18 F]TBAF MeCN 100W 6min 24.4±5.5% ( n  =14) f a Radiolabeling was carried out with a radical scavenger (TEMPO, 1mg). b No TEMPO use. c Solvent (DMF- N  0 ,N  0 -dimethylformamide or MeCN–acetonitrile, 300 l L) was used with H 2 O (10 l L). d No water use. e Progress of the reaction (as shown Scheme 3) and yields were analyzed by radio-TLC (developing solvent: ethyl acetate/hexane=40:60, v/v). f  Yield of isolated pure compounds ([ 18 F] 1–3 ) by semipreparative column using HPLC (55:45 acetonitrile–water, 254nm, 3mL/min). g No product.2982  B. C. Lee et al./Bioorg. Med. Chem. 19 (2011) 2980–2990  expectedto attackthe moreelectrondeficient ringin diaryliodoni-umtosylate precursor, sothat we have preparedvariousdiaryliod-onium salt precursors with the ‘dispensable ring’ is phenyl, or themore electron rich rings;  p -methoxyphenyl,  p -methylphenyl, and2- or 3-thiophenyl on 6-position of benzothiazole ring for [ 18 F] 2 ,which might be superior to fluorine-18 labeling efficiency.The aromatic radiofluorination between a diaryliodonium saltand fluoride is dominated by an electron-rich heteroaromatic ring,while the effects of base and solvent also play a role in increasingthe florine-18 labeling yield. Various conditions were explored forthe preparation of the three BTA analogs [ 18 F] 1 – 3  from differentdiaryliodonium tosylate precursors  12 ,  13a – e  and  14 , whose prep-arationhasbeenpreviouslydescribed(Schemes1and2).Whenthe precursor ( 14 , 2mg scale) was reacted with cesium [ 18 F]fluorideunder general anhydrous radiofluorination conditions, aromaticradiofluorination did not work (Table 1, entry 1). In condition of acetonitrile for solvent or potassium [ 18 F]fluoride Kryptofix 2.2.2for the fluorine-18 salt form, the radiochemical yields were poor(data not shown). Considering that these low radiochemical yieldsmight be due to the instability of iodoniumtosylate, McEwenet al.noted that diaryliodonium salts carried out radical-induceddecomposition in a base condition and generated aromatic hydro-carbon. 34 Therefore, we added a radical scavenger, 2,2,6,6-tetra-methylpiperidine 1-oxyl (TEMPO) to the solvent (300 l L) andfound that the yields were significantly increased, except forDMF solvent (entry 5). 35 Microwave irradiation also successfullyimproved the yields (entries 2 vs 3 and 11 vs 15), as comparedto an oil bath system. In the aqueous environment (addition of 10 l Lofwater),fluorine-18labelingyieldwasincreasedmorethananhydrousconditionincaseofmicrowaveirradiationconditionfor360s in 18–35%yield (entries 7–10 and 15). On the other hand, anoil bath heating in the anhydrous acetonitrile at 130  C for 10mingave higher yield than aqueous environment (entry 11 vs 12).Inordertounderstandtheelectron-richheteroaromaticringef-fect, we prepared five diaryliodonium salt precursors containingthe more electron rich rings;  p -methoxyphenyl,  p -methylphenyl,and 2- or 3-thiophenyl, compare to phenyl ring on 6-position of benzothiazole ring for [ 18 F] 2  (Scheme 2) and evaluated their aro-matic radiofluorination using an oil bath heating. In general, thefluorine-18anionattackedthe more electrondeficient ring, sothatwhen these diaryliodonium salt precursors bearing electron-donating groups (4-MeO and 4-Me) and electron-rich heteroatomaryl (2- or 3-thiophenyl) tend to undergo nucleophilic aromaticfluorination on the contrary benzothiazole ring. These electron-richheteroaromaticringeffect hadanimpact ontheradiofluorina-tion of benzothiazole ring as shown inTable 2. The results indicatethat compounds,  13b  and  13c  did not work effectively compare tocompound  13a , while  para -electron-donating groups substitutedcompounds,  13d  and  13e  showed high aromatic radiochemicalyields of [ 18 F] 2 (Table 2, entries 4 and 5). Consequently, three fluo-rine-18 labeled compounds [ 18 F] 1 – 3  were synthesized with de-sired yields in an environment of radical degeneration,microwave irradiation of 360s or oil bath heating. On the 2-mgscale of precursors in acetonitrile, the radiochemical yields of [ 18 F] 1 – 3  at end-of-synthesis (EOS) were 19–40%, which includedHPLC isolation and a total time of 40–60min (Scheme 3). Theseregioselectivity of diaryliodoniim salt precursors in benzothiazoleringwill beuseful inintroducingno-carrier-addedfluorine-18intoaromatic region of PET probes. 2.2. Determination of   K  i -values The three prepared 6-fluoro-substituted BTA analogs showed  K  i values in the range of 5.5–26.2nM (PIB=5.8nM) on A b 1–42 , indi-cating that fluorine substituted compounds at the 6-position of benzothiazole bind to the same site with good affinity (Table 3), just as known BTA analogs do on AD homogenates despite the factthat the fluorine ion has decreasing electron-donating capacity ascomparedtoahydroxygroupinPIB. 36 Zhengetal.alsosynthesized6-substituted benzothiazole anilines and evaluated their possibili-ties as an amyloid-imaging probes using AD human brain homog-enates(Table3). 14 Althoughtheaffinitiesof theircompoundswereshowntobebelow1nMas measuredusing[ 3 H]-PIB, theyfailedtolabel at the 6-position of their compounds with radioisotopes. 2.3. In vitro fluorescent staining assay  The thioflavine-S dye fluorescence is widelyusedfor the identi-fication of amyloid fibrils in vitro. Our target compounds also have  Table 2 The radiofluorination with diaryliodonium tosylates  13a – e  and hydrolysis a Entry Precursor Yield b (decay-corrected yields) c 1  13a  23.5±7.7% (19.3%) c 2  13b  18.9±8.3%3  13c  26.5±8.1%4  13d  34.3±6.2% (30.1%) c 5  13e  60.4±5.6% (40.5%) ca Radiolabeling of precursors (2mg) were carried out with a radical scavenger(TEMPO, 1mg), acetonitrile solvent without H 2 O at 130  C for 10min using[ 18 F]TBAF. Deprotection was carried out using 3M HCl in ethyl acetate (200 l L) at75  C for 10min. b Progress of the reaction (as shown scheme 3) and yields were analyzed byradio-TLC (developing solvent: ethyl acetate/hexane=40:60, v/v) ( n  =5). c Yieldsofisolatedpurecompound[ 18 F] 2 bysemipreparativecolumnusingHPLC(55:45 acetonitrile–water, 254nm, 3mL/min). +-OTs 12 : R = NO 2 13a-e : R = NCH 3 Boc 14 : R = N(CH 3 ) 2 [ 18 F] 1 : R' = NH 2 [ 18 F] 2 : R' = NHCH 3 [ 18 F] 3 : R' = N(CH 3 ) 2 (i) or (ii) or (iii)19 -40% EOS yieldsSNIRArSN 18 FR' Scheme 3.  Reagents and conditions: (i) (a)  n Bu 4 N[ 18 F]F, CH 3 CN, TEMPO, M.W. (100W), 6min; (b) SnCl 2 , EtOH, 90  C,10min for [ 18 F] 1 ; (ii) (a)  n Bu 4 N[ 18 F]F, CH 3 CN, TEMPO,M.W. (100W), 6min or 130  C, 10min; (b) EtOAc/HCl (v/v=3:1), 75  C, 10min for [ 18 F] 2 ; (iii)  n Bu 4 N[ 18 F]F, CH 3 CN, TEMPO, M.W. (100W), 6min for [ 18 F] 3 .  Table 3 K  i  values of 2-aryl-6-fluorobenzothiazole analogs ( 1–3 ) and log  P   of 2-aryl-6-[ 18 F]fluorobenzothiazole analogs ([ 18 F] 1–3 ) Compound  K  ia (nM) Log P  oct/PBSc 1  26.2 2.83±0.02 2  5.5 (0.2) b 3.20±0.06 3  5.9 (0.3) b 3.60±0.09PIB 5.8 (1.6) b 1.2 da K  i  wasmeasuredby[ 125 I]TZDMcompetitionbindingstudiestoaprecipitate of synthetic A b 1–42  peptide. b Previously published values 16 shown here for comparison ( K  i was measured by radioligand [ 3 H]-PIB competition binding studiesin human brain homogenate). c Values are mean±SD ( n  =5). d Ref. 5. B. C. Lee et al./Bioorg. Med. Chem. 19 (2011) 2980–2990  2983  similar structure, benzothiazole analog, to thioflavine-S and fluo-resce strongly with excitation and emission. As expected, both thi-oflavine-S and compound  2  clearly bind plaque using brainsections of double transgenic mouse (Fig. 2 A and C). Other setsof images (Fig. 2 B and D) indicate that compound  2  has specificbinding to amyloid plaque in mouse brain. 2.4. Partition coefficient measurement In order to measure the potential of compounds [ 18 F] 1 – 3  tocross the blood–brain barrier (BBB) by passive diffusion, the logof the partition coefficient (log P  oct/PBS ) values were distributed inthe range of 2.83–3.60 (Table 3). The results suggested that thethree compounds readily cross the BBB, and that [ 18 F] 1  and[ 18 F] 2 , in particular, are within the optimal range (log P   between1 and 3). 2.5. In vitro stability studies Radiotracers [ 18 F] 1 – 3  were incubated in human serum at 37  Cand analyzed by radio-TLC (data not shown). The results showedthat over 90% of the three radiotracers remained intact even after120min, demonstrating their high chemical stability. 2.6. Analysis of metabolites Radiolabeled materials were extracted from ICR mice brainhomogenates with over 95% efficiency. When samples of brainwere analyzed by HPLC, ratios of peak area under metabolites to[ 18 F] 2  were 5:95 at 5min, 19:81 at 30min, and 42:58 at 60minsample of the brain. 2.7. Biodistribution in normal mice To measure their in vivo brain penetration and clearance, bio-distribution studies of [ 18 F] 1 – 3  were performed in healthy maleICRmice,andtheirblood,remnant, cortexandcerebellumregionalbrain tissue uptakes were determined at different points in time(at2,30, and60min,Table4). Foranideal invivoA b  plaqueprobe,a high initial brain entry and low nonspecific binding in a normalbrain are necessary in order to increase the signal-to-noise ratio.Table 4 lists the tracer uptake in the brain as the most importantterms of percent injected dose per gram of organ (% ID/g).In a comparison of the three radiotracers [ 18 F] 1 – 3 , the initialbrain uptake in the cortex of [ 18 F] 2  was the highest of the threecompounds (6.62±0.3% ID/g at 2min post-injection), and itsradioactivity was rapidly washed out from the brain at 30 and60min post-injection (1.20±0.2 and 0.73±0.1% ID/g, respec-tively). [ 18 F] 1  and [ 18 F] 3  showed an initial uptake in the cortex of 5.86±0.3 and 4.39±0.3% ID/g, respectively. In the measure of brain clearance for the three radiotracers expressed as the ratioof % ID/g in the cortex at 2min over % ID in the cortex at 60min,[ 18 F] 2  showed a higher value (9.1) than [ 18 F] 1  (8.0) and [ 18 F] 3 (2.7). The blood levels of [ 18 F] 1 – 3  were relatively low at all pointsin time. Figure 3 shows the brain radiouptake of [ 18 F] 1 – 3  in ICR mice in terms of percent injected dose per gram of organ normal-ized to body weight (% ID-kg/g) to compare across animals of dif-ferent size. Figure 2.  In vitro fluorescent staining of brain sections from double transgenic (APPswe/PS1 4 E9, 23months) and the wild-type mouse: confocal tile scan images—(A)Thioflavine-Sin a APPswe/PS1 4 E9mousebrain; (B) Thioflavine-Sin a wild-typemousebrain; (C)compound 2 ina APPswe/PS1 4 E9mousebrain; (D) compound 2 ina wild-type mouse brain.  Table 4 Tissue distribution of 2-aryl-6-[ 18 F]fluorobenzothiazoles ([ 18 F] 1–3 ) Radiotracer Time Tissue % ID/g±SDBlood Cortex Cerebellum Remnant a [ 18 F] 1  2min 1.92±0.1 5.86±0.3 5.83±0.2 6.41±0.230min 1.23±0.5 1.79±0.3 2.19±0.3 2.86±0.460min 1.21±0.1 0.73±0.1 1.02±0.2 1.37±0.1[ 18 F] 2  2min 2.08±0.2 6.62±0.3 6.24±0.4 6.30±0.530min 1.13±0.1 1.20±0.2 1.38±0.2 1.91±0.360min 1.11±0.1 0.73±0.1 0.98±0.1 1.32±0.2[ 18 F] 3  2min 1.55±0.1 4.39±0.2 4.45±0.2 4.26±0.230min 1.04±0.2 2.17±0.3 2.03±0.3 2.80±0.460min 1.02±0.1 1.61±0.2 1.16±0.1 2.01±0.2 a Remnant means the remaining brain tissues after extraction of cerebral cortexand cerebellum.2984  B. C. Lee et al./Bioorg. Med. Chem. 19 (2011) 2980–2990
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