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Letter 2c

A potential PET radiotracer for the 5-HT receptor: Synthesis and in vivo evaluation of 4-(3-[ F]fluorophenethoxy)pyrimidine 18

Juhyeon Kim, Byung Seok Moon, Byung Chul Lee, Ho-Young Lee, Hak Joong Kim, Hyunah Choo, Ae Nim Pae, Yong Seo Cho, and Sun-Joon Min ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00445 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 20, 2017

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ACS Chemical Neuroscience

A potential PET radiotracer for the 5-HT2C receptor: Synthesis and in vivo evaluation of 4-(3-[18F]fluorophenethoxy)pyrimidine

Juhyeon Kim,1.2 Byung Seok Moon,3 Byung Chul Lee,3 Ho-Young Lee,3,* Hak-Joong Kim,2 Hyunah Choo,1,4 Ae Nim Pae,4,5 Yong Seo Cho,1,4 Sun-Joon Min,6,*

1

Center for Neuro-Medicine, Korea Institute of Science and Technology (KIST), 5

Hwarangno 14-gil, Seongbuk-gu, Seoul, 02792, Republic of Korea 2

Department of Chemistry, Korea University, Seoul, 02841, Republic of Korea

3

Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam

13620, Republic of Korea 4

Department of Biological Chemistry, Korea University of Science and Technology (UST),

217 Gajungro, Yuseong-gu, Daejeon, 34113, Republic of Korea 5

Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, KIST,

Seoul 02792, Republic of Korea 6

Department of Chemical & Molecular Engineering/Applied Chemistry, Hanyang University,

Ansan, Gyeonggi-do, 15588, Republic of Korea.

*To whom correspondence should be addressed: Sun-Joon Min, Ph.D., Department of Chemical & Molecular Engineering/Applied Chemistry, Hanyang University, 55 Hanyangdaehak-ro, Sangnok, Ansan, Gyeonggi-do 15588, Republic of Korea (P) +82-31-400-5502/(F) +82-31-400-5457; Email: [email protected] Ho-Young Lee, M.D., Department of Nuclear Medicine, Seoul National University Buandang Hospital, 82 Gumi-ro 173, Bundang, Seongnam, Gyeonggi-do, 13620, Republic of Korea; Email: [email protected]

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Abstract: The serotonin 2C receptor subtype (5-HT2C) is an excitatory 5-HT receptor widely distributed throughout the central nerve system. As the 5-HT2C receptor displays multiple actions on various neurotransmitter systems including glutamate, dopamine, epinephrine and γ-aminobutyric acid (GABA), abnormalities of the 5-HT2C receptor are associated with psychiatric diseases such as depression, schizophrenia, drug abuse, and anxiety. Up to date, three kinds of 5-HT2C PET radiotracers such as [11C]N-methylated arylazepine (1), [11C]WAY-163909 (2), [18F]fluorophenylcyclopropane (3) have been developed, but they may not be suitable for in vivo 5-HT2C imaging study due to their modest specific binding. Herein, the synthesis and in vivo evaluation of 4-(3-[18F]fluorophenethoxy)pyrimidine [18F]4 as a potential PET radiotracer for the 5-HT2C receptor is described. [18F]4 was synthesized by nucleophilic aromatic substitution of diaryliodonium precursor 17a with a 7.8 ± 2.7% (n = 6, decay corrected) radiochemical yield and over 99% radiochemical purity, showing an 89 ± 14 GBq/µmol specific radioactivity. The in vivo PET imaging studies of [18F]4 with or without locaserin, an FDA approved selective 5-HT2C agonist, demonstrated that [18F]4 exhibits a high level of specific binding to 5-HT2C receptors in the rat brain.

Keywords: 5-HT2C receptor, in vivo, PET radioligands, brain imaging, psychiatric disorders

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GRAPHICAL TABLE OF CONTENTS OTs I

N O F

N

N N

18 Boc 1) nBu4[ F]F, TEAB, TEMPO, 130 °C, 10 min

RCY = 7.8±2.7% S.A. = 89±14 GBq/ µmol

With blocking

18

F

NH O

2) 2N HCl, 100 °C, 10 min

F

N

N N

Without blocking

2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter, is a key modulator of various biological functions in the central nervous system (CNS). It acts as an endogenous ligand to activate serotonin receptors that mediate a number of neurological processes including regulation of mood and sleep, control of appetite, learning and memory, and cognitive performance.1-4 There are 14 distinct 5-HT receptor subtypes, most of which belong to a group of G-protein coupled receptors (GPCR). On the basis of their structural homology, amino acid sequence, and signalling pathway, they are categorized into one of seven 5-HT classes (5-HT1-7). The 5-HT2C receptor is an excitatory 5-HT receptor widely distributed throughout the CNS. 5-HT2C receptors are highly localized in the choroid plexus and are moderately observed in the hypothalamus, globus pallidus, and substantia nigra, whereas lower densities of this receptors are found in the cortex and cerebellum.5 Furthermore, the pharmacological profiles of the 5-HT2C receptor are very similar in human, pig and rat on the basis of both radioligands and autoradiographic procedures in frontal cortex, hippocampus, and choroidal plexus of these species using [3H]mesulergine.6-8 As the 5-HT2C receptor displays multiple actions on various neurotransmitters and receptors, abnormalities of 5-HT2C receptors are associated with psychiatric diseases such as depression, schizophrenia, drug abuse, and anxiety.9-15 Although a number of 5-HT2C modulators have been developed as chemical tools for the pharmacological study of the 5-HT2C receptor,16-18 it is difficult to elucidate the role of the 5-HT2C receptor in diseases due to the lack of suitable methods for determining the 5-HT2C concentration in vivo.

Figure 1. Examples of 5-HT2C radioligands for PET molecular imaging

3


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Positron emission tomography (PET), one of the non-invasive imaging technologies that measures biochemical processes in a living system, is often used for clinical research and drug development. In particular, PET is useful for tracking a specific neurotransmitter in regions of brain and for measuring the occupancy of therapeutic drugs in receptors or transporters. While PET radioligands have been developed for the imaging of several 5-HT receptor subtypes to evaluate their function in physiology and psychiatric disorders, there are no readily available radiotracers for in vivo imaging of the 5-HT2C receptor.19-21 Recently, three 5-HT2C PET tracers have been developed based on potent and selective

5-HT2C

agonists

as shown in Figure 1.22-24 Compounds 1 and 2 were synthesized using efficient radiolabelling processes with different carbon-11 sources such as methyl iodide and formaldehyde, but their preliminary PET imaging data in animal models represent non-specific binding. [18F]-labelled compound 3, synthesized via metal-catalysed arene radiofluorination, showed excellent brain penetration and metabolic stability. However, it is not suitable for in vivo 5-HT2C imaging due to its modest specific binding. Thus, new 5-HT2C radiotracers with improved specific binding in vivo are required for understanding the full potential of the serotonergic system in psychiatric disorders. Among reported 5-HT2C agonists,25-28 we selected pyrimidine analogue 4 as a potential target molecule for 5-HT2C radioligand discovery due to its pharmacological and pharmacokinetic properties.29 It shows good metabolic stability and low in vitro toxicity as well as reasonable selectivity over other 5-HT subtypes. In particular, it is reported that 4 did not show any significant agonistic effect on the 5-HT2A receptor (EC50 of 5-HT2C = 1.7 nM, Emax = 88% vs EC50 of 5-HT2A = 4441 nM, Emax = 62%),29 which is crucial for imaging a brain region of high 5-HT2C population. With regard to radiochemical synthesis, we assumed that the position of the fluorine substituent on the aryl group of compound 4 would be amenable to radiolabelling with [18F]-fluoride ion by nucleophilic aromatic substitution (SNAr). Thus, we 4



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considered aryliodonium salt 5 as a crucial precursor for radiofluorination, which might be accessible via a ligand exchange of hypervalent iodine with the corresponding arylstannane 6 (Scheme 1).30-38 In the current study, we report an efficient synthesis of [18F]-labelled pyrimidine 4 and in vivo evaluation of this compound as a 5-HT2C PET radioligand for brain imaging.

Scheme 1. Target molecule 4 and a synthetic design for its radiofluorination Initially, we synthesized pyrimidine analogue 4 according to the procedure reported in the literature,29 and verified its binding affinities to 5-HT subtypes. The assay results showed that 4 has high binding affinities to 5-HT2A, 5-HT2B, 5-HT2C, 5-HT6 and 5-HT7 with >90% binding at a concentration of 10 µM. (Table 1). Further determination of Ki values against 5HT receptor subtypes indicated that 4 displayed the highest binding affinity to 5-HT2C as anticipated. Most importantly, we observed at least 11-fold selectivity for 5-HT2C over other subtypes, which confirmed that 4 has an excellent binding affinity profile for development as a PET radioligand. Table 1. Binding affinity evaluation of compounds 4 against 5-HT receptor subtypesa 5-HT subtypes % binding at 10 µM Ki (nM)

1A

1B

1D

1E

2A

2B

2C

3

5A

6

7

89.0

55.1

78.3

65.6

95.6

97.4

98.2

75.0

37.4

93.8

94.7

353.0

1780.0

542.0

1160.0

128.0

7.9

0.7

241.0

NDb

43.0

84.0

a

3

5-HT receptor binding was determined by competitive binding assay using [ H]-labelled compound as a radioligand for the specific subtype (see Table S3). b Not determined

5


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Scheme 2. Synthesis of arylstannane 6. Reagents and conditions: (a) (R)-(+)-propylene oxide, CuI, THF, rt, 78%; (b) 2,4-dichloro-5-fluoropyrimidine, NaOt-Bu, toluene, rt, 91%; (c) (R)(+)-1-Boc-3-methylpiperazine, K2CO3, DMSO, 130 °C, 52%; (d) BBr3, DCM, rt, 71%; (e) (Boc)2O, NaHCO3, dioxane, H2O, 0 °C to rt, 83%; (f) Tf2O, DIPEA, DCM, 0 °C, 91%; (g) Pd(PPh3)4, bis(tributyltin), LiCl, DMF, 130 °C, 63%.

As described in Scheme 2, the synthesis of arylstannane 6 commenced with formation of secondary alcohol 9 by Grignard reaction of 8 with optically active propylene oxide. The SNAr reaction of 9 with 2,4-dichloro-5-fluoropyrimidine selectively afforded pyrimidine 10 in 91% yield, which was subjected to a second SNAr reaction with N-Boc-piperazine to give highly substituted pyrimidine 11. Treatment of 11 with boron tribromide easily removed the phenolic methyl group, but with concomitant cleavage of the Boc group. After reinstallation of the Boc protecting group, phenol 13 was converted to triflate 14. As formation of the desired arylstannane 6 using the Stille reaction was somewhat problematic, optimization of the reaction condition for this process were investigated (Table S1). The highest yield for this 6



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conversion was achieved when the reaction of 14 with dibutyltin in DMF was performed in the presence of Pd(PPh3)4 and LiCl at 130 °C.39 Next, we prepared diaryliodonium tosylate 17 as a radioligand precursor and investigated fluorination reactions applicable to radiolabelling (Scheme 3). In general, aromatic fluorination of diaryliodonium salts with fluoride ion occurs at the least electron-rich ring. Thus, four different kinds of diaryliodonium salts 17a-d were synthesized with a modified version of the literature procedure.40-43 First, the aryl iodides were converted to aryliodo diacetates 15a-d by oxidation with either NaBO3 or NaIO4 under acidic conditions. Then, treatment of 15a-d with TsOH generated iodine (III) intermediate 16a-d, which underwent ligand exchange with 6 to afford the corresponding iodonium salts 17a-d in good yields. Finally, we investigated fluorination reactions of 17a-d using CsF as a fluoride ion source. Indeed, all of the reactions produced the desired compound 4 along with side-product 18 or 19. (Table S2). Although compounds 17b-d consist of the more electron-rich rings, we obtained the best yield of 4 when 17a was used as a substrate. Thus, the reaction of 17a with CsF at 90 °C for 10 min followed by treatment of TFA for 10 min afforded a 1.8:1 mixture of 4 and 18 in moderate yield, which was separable by HPLC within 30 min. It should be noted that the fluoride ion was preferentially incorporated to the desired phenyl ring although both aryl rings of 17a have similar electronic character.

7


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OAc I Ar OAc

a or b Ar

I

OH I OTs Ar

c

16a-d

15a (Ar = toluene) 15b (Ar = anisole) 15c (Ar = 2-thiophene) 15d (Ar = 3-thiophene)

d

OTs R

N O

H

N

N

Ar

I

N

e,f

O

N

F

N

N N

F

4 (R = F) 18 (R = I) 19 (R = H)

Boc

17a-d

Scheme 3. Synthesis and fluorination of diaryliodonium tosylate 17. Reagents and conditions: (a) NaBO3, AcOH, 50 °C, 30-47%; (b) NaIO4, NaOAc, AcOH, Ac2O, 120 °C, 91%; (c) TsOH-H2O, CH3CN, 0 °C; (d) 6, CHCl3, reflux (100−120 °C), 50-79%; (e) CsF, DMF, 90 °C, 53% (from 17a); (f) TFA, CH2Cl2, rt, 53% (4:18 = 1.8:1) . Having established the synthetic protocol for fluorination, we next prepared

18

F-labeled 4

starting from tosylate 17a via the SNAr reaction. Although CsF was used as the fluoride ion source during our optimization process, reproducible results were obtained when the radiofluorination

reaction

was

performed

using

tetrabutylammonium

[18F]fluoride

(nBu4N[18F]F) as shown in Scheme 4. Thus, a solution of 17a and nBu4N[18F]F in DMF was heated in the presence of TEMPO as a radical scavenger at 130 °C for 10 min and the BOC protecting group was subsequently removed using 2 N aqueous hydrochloric acid. The resulting crude mixture was purified by reverse phase HPLC with a guard column to afford the desired [18F]4, which was reformulated in 10% ethanol/saline solution with a tC18 Seppak cartridge. Consequently, the isolated radiochemical yield was 7.8 ± 2.7% (n = 6, decay corrected) with higher than 99% radiochemical purity. The total operation time including HPLC purification was 110 ± 4 min, and the specific radioactivity was 89 ± 14 GBq/µmol at the end of the synthesis. 8



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Scheme 4. Radiofluorination of diaryliodonium tosylate 17a. Reagents and conditions: (a) nBu4N[18F]F, TBAHCO3, TEMPO, DMF, 130 °C, 10 min; (b) 2N HCl, CH3CN/H2O (3:1), 100 °C, 10 min. Finally, [18F]4 was intravenously administered to normal rats through the lateral tail vein and brain PET/CT images were acquired immediately after injection in 3D dynamic acquisition mode with a micro PET/CT scanner (nanoScan PET/CT, Mediso Inc., Budapest, Hungary). We observed that [18F]4 exhibited excellent brain blood barrier penetration (Figure 2A and 2B). The regional uptake of [18F]4 within the brain showed high uptake in the thalamus, striatum, frontal cortex, and choroid plexus, whereas the cerebellum, a region known to have a low density of 5-HT2C receptors, showed the lowest uptake. Moreover, time−activity curves for several regions of interest indicated nearly identical pharmacokinetics throughout the brain. In order to verify whether [18F]4 showed specific binding to 5-HT2C receptors, [18F]4 was co-administrated with cold compound 4 or locaserin, an FDA approved selective 5-HT2C agonist (7.5-fold higher binding affinity for 5-HT2C over 5-HT2A44) (Figure 2D and 2F). Although the off-target effect of locaserin with excess of 3 mg/kg has been reported,45 the same dose (10 mg/kg) of compound 4 or locaserin was intravenously injected for the blocking experiments. The uptake of [18F]4 in the thalamus, striatum, frontal cortex and choroid plexus was decreased after co-treatment with compound 4 (Figure 2C). Furthermore, we observed reduced uptake in the same brain region, but no change in the cerebellum after blocking with locacerin (Figure 2E). These results suggest that the PET images acquired with [18F]4 represent the specific binding to 5-HT2C receptors. 9


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(A)

(B)

(C)

(D)

(E)

(F)

18

[ F]4

18

[ F]4 + cold 4

18

[ F]4 + locaserin

Figure 2. PET analysis of [18F]4 in normal rats. (A) Summation image of [18F]4 dynamic PET/CT data. (B) Time-activity curves for brain regions of interest in [18F]4 study. (C) Summation image of [18F]4 dynamic PET/CT data with co-treatment with cold compound 4. (D) Time-activity curves for brain regions after co-treatment with cold compound 4. (E) Summation image of [18F]4 dynamic PET/CT data with co-treatment with locaserin. (F) Time-activity curves for brain regions after co-treatment with locaserin. The PET images shown were acquired using same rats. 10



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In conclusion, we successfully synthesized a 4-(3-[18F]fluorophenethoxy)pyrimidine as a potential PET radiotracer for 5-HT2C receptors via the SNAr reaction. Use of (4methylphenyl)iodonium tosylates 17a as a precursor was crucial for introduction of [18F]fluoride ion into the 3 position of the phenethyl moiety. In an in vivo evaluation of [18F]4 in rats, [18F]4 exhibited high brain uptake with high specific binding to 5-HT2C receptors. Therefore, [18F]4 is a suitable candidate for in vivo 5-HT2C PET imaging, which will be further studied for clinical development.

METHODS

General: All reactions were carried out under dry nitrogen unless otherwise indicated. Commercially available reagents were used without further purification. Solvents and gases were dried according to standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator. Analytical thin layer chromatography (TLC) was performed using glass plates precoated with silica gel (0.25 mm). TLC plates were visualized by exposure to UV light (UV), and then were visualized with a p-anisaldehyde stain followed by brief heating on hot plate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents. 1H and 13C spectra were recorded on Bruker 300, Bruker 400 or Varian 300 NMR spectrometers. 1H NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constant (J) in Hertz (Hz). 1H NMR chemical shifts are reported relative to CDCl3 (7.26 ppm). 13C NMR was recorded relative to the central line of CDCl3 (77.0 ppm). LC/MS analyses were performed on Shimadzu LCMS-2020 system. Synthesis of intermediates and precursor. (R)-2-Chloro-5-fluoro-4-((1-(3-methoxyphenyl) propan-2-yl)oxy)pyrimidine (10). To a solution of sodium tert-butoxide (927 mg, 9.65 11


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mmol) in toluene (18.2 mL) was treated with alcohol 9 (802 mg, 4.82 mmol) dropwise at 0 °C. After 5 min, 2,4-dichloro-5-fluoropyrimidine (967 mg, 5.79 mmol) was added to the mixture. The reaction mixture was allowed to stir at the room temperature for 1 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with EtOAc and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:8) to afford pyrimidine 10 (1.30 g, 91%) as a colorless oil; Rf = 0.60 (EtOAc:n-hexane = 1:4). 1H-NMR (400 MHz, CDCl3) δ 8.13 (d, J = 2.3 Hz, 1H), 7.21-7.18 (m, 1H), 6.83-6.82 (m, 2H), 6.78-6.75 (m, 1H), 5.57-5.48 (m, 1H), 3.79 (s, 3H), 3.08-2.85 (m, 2H), 1.42 (d, J = 6.2 Hz, 3H).

13

C-NMR (100 MHz,

CDCl3) δ 160.1, 159.5 (d, 2J = 11 Hz), 153.7 (d, 4J = 5 Hz), 146.5 (d, 1J = 252 Hz), 144.4 (d, 2

J = 20 Hz), 138.9, 130.0, 122.3, 115.6, 112.7, 76.8, 55.6, 42.7, 19.7. HRMS-ESI (m/z): [M +

Na]+ calcd for C14H14ClFN2NaO2: 319.0620; found: 319.0622. Optical rotation for (R)-10: [α]D 23 = -18.6 (c = 0.234, CHCl3). (R)-tert-Butyl-4-(5-fluoro-4-(((R)-1-(3-methoxyphenyl)propan-2-yl)oxy)pyrimidin-2-yl)3-methylpiperazine-1-carboxylate (11). To a solution of pyrimidine 10 (631 mg, 2.13 mmol) in DMSO (21.3 mL) was added (R)-(+)-1-Boc-3-methyl piperazine (511 mg, 2.55 mmol) and potassium carbonate (353 mg, 2.55 mmol). The reaction mixture was allowed to stir at 130 °C for 15 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with EtOAc and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford methyl piperazine carboxylate 11 (513 mg, 52%) as a colorless oil; Rf = 0.33 (EtOAc/nhexane = 1:4). 1H-NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.9 Hz, 1H), 7.18-7.14 (m, 1H), 6.79-6.72 (m, 3H), 5.43-5.35 (m,1H), 4.68 (s, 1H) 4.24 (d, J = 13.1 Hz, 1H), 4.11-3.87(m, 12



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2H), 3.75 (s, 3H), 3.13-3.06 (m, 3H) 2.82-2.77 (m, 2H), 1.45 (s, 9H), 1.33 (d, J = 6.2 Hz, 3H), 1.11 (d, J = 6.7 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 160.5, 158.6 (d, 2J = 11 Hz), 157.8 (d, 4J = 2 Hz), 156.2, 144.0 (d, 2J = 30 Hz), 141.0 (d, 1J = 247 Hz), 140.1, 130.2, 122.7, 116.1, 112.8, 80.7, 74.6, 56.0, 49.5, 48.1, 44.9, 43.9, 43.2, 39.7, 29.3, 20.3, 15.0. HRMS-ESI (m/z): [M + Na]+ calcd for C24H33ClFN4NaO4: 483.2378; found: 483.2378. Optical rotation for (R)11: [α]D 23 = 48.3 (c = 0.323, CHCl3). 3-((R)-2-((5-Fluoro-2-((R)-2-methylpiperazin-1-yl)pyrimidin-4-yl)oxy)propyl)phenol (12) To a solution of methyl piperazine carboxylate 11 (401 mg, 0.870 mmol) in CH2Cl2 (43.5 mL) was added boron tribromide (873 mg, 3.48 mmol) dropwise at -78 °C. The reaction mixture was allowed to stir at 0 °C for 2 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NaHCO3, extracted with CH2Cl2 and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM/MeOH = 10:1) to afford methyl piperazine 12 (215 mg, 71%) as a colorless oil; Rf = 0.28 (DCM/MeOH = 10:1). 1

H-NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.8 Hz, 1H), 7.09-7.05 (m, 1H), 6.73-6.64 (m, 3H),

5.48-5.40 (m, 1H), 4.81-4.79 (m, 1H), 4.37 (d, J = 12.0 Hz, 1H), 3.30 (d, J = 12.3 Hz, 1H), 3.22-3.03 (m, 4H), 2.87-2.74 (m, 2H) , 1.35 (d, J = 6.2 Hz, 3H), 1.30 (d, J = 6.9 Hz, 3H). 13CNMR (100 MHz, CDCl3) δ 159.1 (d, 2J = 11 Hz), 157.47, 157.4 (d, 4J = 3 Hz), 144.2 (d, 2J = 20 Hz), 141.4 (d, 1J = 247 Hz), 140.6, 130.7, 122.6, 117.5, 114.9, 75.0, 49.8, 46.5, 45.7, 43.6, 38.8, 20.9, 15.2. HRMS-ESI (m/z): [M + H]+ calcd for C18H24FN4O2: 347.1878; found: 347.1880. Optical rotation for (R)-12: [α]D 23 = -51.7 (c = 0.118, CHCl3). (R)-tert-Butyl-4-(5-fluoro-4-(((R)-1-(3-hydroxyphenyl)propan-2-yl)oxy)pyrimidin-2-yl)3-methyl piperazine-1-carboxylate (13). To a solution of methyl piperazine 12 (31.0 mg, 0.0890mmol) in H2O:Dioxane=1:1 (1.80 mL) cooled to 0 °C was added sodium bicarbonate 13


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(8.23 mg, 0.0980 mmol) and di-t-butyldicarbonate (22.0 mg, 0.0980 mmol). The reaction mixture was allowed to stir at the room temperature for 1 h. After completion of the reaction (monitored by TLC), the mixture was diluted with saturated aqueous NaHCO3 and extracted with CH2Cl2. The organic layers were dried over anhydrous MgSO4 and concentrated in

vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM/MeOH = 10:1) to afford phenol 13 (33.0 mg, 83%) as a colorless oil; Rf = 0.59 (DCM/MeOH = 10:1). 1

H-NMR (400 MHz, CDCl3) δ 7.94 (d, J = 3.0 Hz, 1H), 7.16-7.12 (m, 1H), 6.79-6.68 (m, 3H),

5.44-5.35 (m, 1H), 4.70 (s, 1H), 4.26 (d, J = 13.4 Hz, 1H), 4.13-3.87 (m, 2H), 3.17-3.06 (m, 3H), 2.93-2.78 (m, 2H), 1.49 (s, 9H), 1.36 (d, J = 6.2 Hz, 3H), 1.15 (d, J = 6.7 Hz, 3H). 13CNMR (100 MHz, CDCl3) δ 158.6 (d, 2J = 3 Hz), 157.4 (d, 4J = 3 Hz), 156.8, 156.1, 143.6 (d, 2

J = 21 Hz), 140.8 (d, 1J = 247 Hz), 140.1, 130.2, 122.2, 117.2, 114.3, 80.9, 74.5, 48.0, 44.7,

43.7, 42.9, 39.5, 29.2, 20.1, 14.9. HRMS-ESI (m/z): [M + Na]+ calcd for C23H31FN4NaO4: 469.2222; found: 469.2220. Optical rotation for (R)-13: [α]D 24 = 97.9 (c = 0.286, CHCl3). (R)-tert-Butyl-4-(5-fluoro-4-(((R)-1-(3-(((trifluoromethyl)sulfonyl)oxy) phenyl)propan-2yl)oxy) pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (14). To a solution of phenol 13 (54.0 mg, 0.120 mmol) in CH2Cl2 (1.20 mL) was treated with N,N-diisopropylethylamine (31.0 mg, 0.240 mmol). After then, trifluoromethane sulfonic anhydride (41.0 mg, 0.150 mmol) was slowly added to the mixture at 0 °C. The reaction mixture was allowed to stir at the room temperature for 0.5 h. After completion of the reaction (monitored by TLC), it was quenched with saturated aqueous NH4Cl, extracted with CH2Cl2 and washed with brine. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:4) to afford triflate 14 (64.0 mg, 91%) as a colorless oil; Rf = 0.41 (EtOAc:n-hexane = 1:4). 1HNMR (400 MHz, CDCl3) δ 7.95 (d, J = 2.9 Hz, 1H), 7.38-7.34 (m, 1H), 7.27-7.25 (m, 2H), 14



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7.17-7.12 (m, 1H), 5.60-5.38 (m, 1H), 4.76-4.68 (m, 1H), 4.27-3.91 (m, 3H), 3.17-3.09 (m, 3H), 3.01-2.87 (m, 2H), 1.48 (s, 9H), 1.37 (d, J = 6.2 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H). 13CNMR (100 MHz, CDCl3) δ 157.4 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 149.6, 143.4 (d, 2

J = 20 Hz), 140.6, 139.9 (d, 1J = 247 Hz), 130.1, 129.6, 122.2, 119.4, 118.7 (q, J = 323 Hz),

79.8, 72.8, 48.4, 47.2, 43.9, 42.8, 41.8, 38.8, 28.4, 19.4, 14.0. HRMS-ESI (m/z): [M + Na]+ calcd for C24H30F4N4NaO6S: 601.1714 ; found: 601.1713. Optical rotation for (R)-14: [α]D 24 = -6.1 (c = 0.245, CHCl3). (R)-tert-Butyl-4-(5-fluoro-4-(((R)-1-(3-(tributylstannyl)phenyl)propan-2-yl)oxy) pyrimidin-2-yl)-3-methylpiperazine-1-carboxylate (6). To a solution of triflate 14 (842 mg, 1.46 mmol) in DMF (4.85 mL) was treated with LiCl (123 mg, 2.91 mmol), tetrakis triphenyl phosphine palladium (168 mg, 0.150 mmol) and tributyltin (2.20 mL, 4.37 mmol). The reaction mixture was allowed to stir at 130 °C for 12 h. After completion of the reaction (monitored by TLC), the mixture was filtered with silica gel and celite pad, extracted with ether. The organic layers were dried over anhydrous MgSO4 and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (EtOAc:nhexane = 1:8) to afford arylstannane 6 (662 mg, 63%) as a colorless oil; Rf = 0.71 (EtOAc:nhexane = 1:4). 1H-NMR (400 MHz, CDCl3) δ 7.93 (d, J = 2.9 Hz, 1H), 7.31-7.22 (m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 5.44-5.36 (m, 1H), 4.69 (s, 1H), 4.27-4.24 (d, J = 2.9 Hz , 1H), 4.142.89 (m, 2H), 3.15-3.08 (m, 3H), 2.86-2.81 (m, 2H), 1.53-1.49 (m, 6H), 1.47 (s, 9H), 1.361.28 (m, 9H), 1.13 (d, J = 6.7 Hz, 3H) 1.02 (t, J =8.1 Hz, 6H), 0.86 (t, J = 7.3 Hz, 9H). 13CNMR (100 MHz, CDCl3) δ 157.7 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 143.1 (d, 2J = 20 Hz), 142.1, 140.1 (d, 1J = 247 Hz), 137.7, 136.9, 134.6, 129.1, 127.9, 79.8, 73.8, 48.5, 47.2, 44.1, 42.9, 42.3, 38.8, 29.1, 28.4, 27.3, 19.3, 14.1, 13.7, 9.5. HRMS-ESI (m/z): [M + Na]+ calcd for C35H57FN4NaO3Sn: 743.3336 ; found: 743.3334. Optical rotation for (R)-6: [α]D 24 = -6.5 (c = 0.123, CHCl3). 15


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(3-((R)-2-((2-((R)-4-(tert-Butoxycarbonyl)-2-methylpiperazin-1-yl)-5-fluoropyrimidin-4yl)oxy) propyl)phenyl)(p-tolyl)iodonium 4-methylbenzene sulfonate (17a). To a solution of diacetate 15a46 (9.30 mg, 0.0490 mmol) in CH3CN (0.250 mL) was added p-TsOH-H2O (16.0 mg, 0.0490 mmol) at 0 °C. The reaction mixture was allowed to stir until the solid disappears. After chloroform (2.50 mL) was added to the mixture, arylstanane 6 (31.9 mg, 0.0440 mmol) was then slowly added. The reaction mixture was allowed to stir at 100 °C for 12 h. After completion of the reaction (monitored by TLC), the mixture was concentrated in

vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM/MeOH = 10:1) to afford iodonium salt 17a (22.5 mg, 63%) as a white solid; Rf = 0.46 (DCM/MeOH = 10:1). 1H-NMR (400 MHz, CDCl3) δ 7.93 (d, J = 2.8Hz, 1H), 7.83 (s, 1H), 7.78-7.74 (m, 3H), 7.66 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.2 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 5.36-5.31 (m, 1H), 4.66 (d, J = 3.9 Hz, 1H), 4.26-3.87 (m, 2H), 3.14-2.93 (m, 5H), 2.38 (s, 3H), 2.32 (s, 3H), 1.48 (s, 9H), 1.34 (d, J = 6.2 Hz, 3H), 1.12 (d, J = 6.4 Hz, 3H).

13

C-NMR (100 MHz, CDCl3) δ 157.3 (d, 2J = 11 Hz),

156.8 (d, 4J = 2 Hz), 155.2, 143.3 (d, 2J = 19 Hz), 142.69, 142.65, 141.8, 139.9 (d, 1J = 247 Hz), 139.4, 135.9, 135.1, 132.9, 132.8, 132.5, 131.4, 128.5, 126.0, 115.3, 111.5, 79.9, 72.7, 48.5, 47.2, 44.0, 43.0, 41.6, 38.8, 28.4, 21.4, 21.3, 19.4, 14.1. HRMS-ESI (m/z): [M + Na]+ calcd for C30H37FIN4O3: 647.1889 ; found: 647.1877. Optical rotation for (R)-17a: [α]D 24 = 164.1 (c = 0.156, CHCl3). Serotonin receptor binding affinity assays. Eleven dilutions (5 x assay concentration) of the test and reference compounds (Table S3) were prepared in standard binding buffer (50 mM

tris(hydroxymethyl)-aminomethane–HCl

(Tris-HCl),

10 mM

MgCl2,

1 mM

ethylenediaminetetraacetate (EDTA), pH 7.4) by serial dilution: 0.05 nM, 0.5 nM, 1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM, 500 nM, 1.5 µM, 5 µM, and 50 µM. The radioligand (Table S3) was diluted to five times the assay concentration in standard binding buffer. Aliquots (50 mL) 16



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of the radioligand were dispensed into the wells of a 96-well plate containing 100 ml of standard binding buffer. Triplicate aliquots (50 mL) of the test and reference compound dilutions were then added. Finally, crude membrane fractions (50 mL) of cells (HEK293 or CHO) expressing human recombinant receptors were dispensed into each well. Total 250 ml of the reaction mixtures were incubated at room temperature and shielded from light for 1.5 h, then harvested by rapid filtration onto Whatman GF/B glass fiber filters presoaked with 0.3% polyethyleneimine, by using a 96-well Brandel harvester. Four rapid washes were performed with chilled standard binding buffer (500 mL) to decrease nonspecific binding. Filters were placed in 6 mL scintillation tubes and allowed to dry overnight. The next day, 4-ml of EcoScint scintillation cocktail (National Diagnostics) was added to each tube. The tubes were capped, labeled, and counted by liquid scintillation counting. The filter mats were dried, and the scintillant was melted onto the filters, then the radioactivity retained on the filters was counted in a Microbeta scintillation counter. The IC50 values were obtained by using the Prism 4.0 program (GraphPad Software) and converted into Ki values. Each compound was tested in triplicate at least. Procedure for radiofluorination. [18F]Fluoride, produced in a cyclotron, was loaded into Chromafix® PS-HCO3 cartridge and eluted with methanol/water (1 mL/0.1 mL) containing tetrabutylammonium bicarbonate (40 wt%, 2 µL).47 The azeotropic distillation of the eluted solution were carried out to afford the corresponding tetrabutylammonium fluoride, to which were added compound 17a (2.5 mg), DMF (0.4 mL) and TEMPO (1 mg). The reaction mixture was heated in oil bath at 130 °C for 10 min, cooled down to room temperature, and diluted with water (10 mL). The resulting solution was loaded into tC18 Sep-Pak cartridge, washed with water (10 mL) and eluted with CH3CN (1.5 mL). The eluted solution was treated with 2 N HCl (0.5 mL), heated at 100 °C for additional 10 min, and then cooled down to room temperature. The reaction mixture was diluted with water (10 mL) and loaded into 17


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tC18 Sep-Pak cartridge, followed by water (10 mL). The crude compounds were collected with CH3CN (1.5 mL) and the eluted solution was injected into HPLC system (Waters, Xterra MS, 10×250 mm). The HPLC purification was performed in 40% CH3CN/aq. NH4OAc (0.1 M) at a flow rate of 3.0 mL/min, using a UV detector at 254 nm and a γ-ray detector in the module. The fraction of [18F]4 collected from HPLC at around 14 min was transferred to a diluted vial with which was filled with water (10 mL) (Figure S1). The diluted solution was exchanged to 10% ethanol-saline solution by tC18 Sep-Pak cartridge to remove the clinically unavailable HPLC solvent. The isolated radiochemical yield was 7.8 ± 2.7 % (n = 6, decay corrected) and produced approximately 1.7-2.2 GBq per batch. An aliquot of the formulated solution was checked by analytical HPLC (Waters, Xterra RP-18, 5 µm, 3.9 x 250 mm; eluent: 70% CH3CN/H2O; flow rate: 1 mL/min) for radiochemical identity, radiochemical purity and specific activity (Figure S2). The radiochemical purity was over 99% and specific radioactivity was 89 ± 14 GBq/µmol at the end of synthesis. In vivo PET imaging acquisition. Animals were positioned prone in the standard rat bed. 1.0 mCi of [18F]4 was intravenously injected via the tail vein, and Brain PET/CT image was acquired right after injection on 3D dynamic acquisition list mode with micro PET/CT scanner until 90 minutes after injection (nanoScan PET/CT, Mediso Inc., Budapest, Hungary). Animal was maintained under anesthesia for the entire duration of the scan with isoflurane (2.5% flow rate). For CT image acquisition, X-ray source was set to a 200 uA and 45 kVp with 0.5 mm. The CT images were reconstructed using conebeam reconstruction with a Shepp filter with a cutoff at the Nyquist frequency and a binning factor of 4, resulting in an image matrix of 480 × 480 × 632 and a voxel size of 125 um. The PET images were reconstructed using Tear-Tomo Real 3D PET engine (nanoScan PET/CT, Mediso Inc., Budapest, Hugary). To evaluate specific binding to 5-HT2C receptor, 10 mg/kg of locaserin or cold form 4 was coincidentally injected with 1.0 mCi of [18F]4. Images were acquired right 18



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after injection on 3D dynamic acquisition list mode until 90 minutes after injection. For the image analysis, each rat brain image was spatially normalized to the rat brain template, and the volume of interest of each region was used for the quantification analysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: 82-31-400-5457. Tel: 82-31-400-5502. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding This work was supported by the Korea Health Industry Development Institute (KHIDI, HI16C1677) and the National Research Foundation (NRF-2016R1A2B1012277 and NRF2014M3C1A3054141). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS Binding affinity data were generously provided by the US National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program (HHSN-271-2008-00025-C). Animal 19


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experimentation was performed according to Guidelines for Accommodation and Care of Animals.

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