A Potential PET Radiotracer for the 5-HT2C Receptor: Synthesis and in Vivo Evaluation of 4-(3-[18F]fluorophenethoxy)pyrimidine

Article abstract of DOI:10.1021/acschemneuro.6b00445

The serotonin 2C receptor subtype (5-HT_2C) is an excitatory 5-HT receptor widely distributed throughout the central nervous system. As the 5-HT_2C receptor displays multiple actions on various neurotransmitter systems including glutamate, dopamine, epinephrine, and γ-aminobutyric acid (GABA), abnormalities of the 5-HT_2C receptor are associated with psychiatric diseases such as depression, schizophrenia, drug abuse, and anxiety. Up to date, three kinds of 5-HT_2C PET radiotracers such as [¹¹C]N-methylated arylazepine (1), [¹¹C]WAY-163909 (2), and [¹⁸F]fluorophenylcyclopropane (3) have been developed, but they may not be suitable for in vivo 5-HT_2C 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-HT_2C 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 lorcaserin, a U.S. Food and Drug Administration approved selective 5-HT_2C agonist, demonstrated that [¹⁸F]4 exhibits a high level of specific binding to 5-HT_2C receptors in the rat brain.

Full text of DOI:10.1021/acschemneuro.6b00445

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Letter
2c
A potential PET radiotracer for the 5-HT receptor: Synthesis
18
and in vivo evaluation of 4-(3-[F]fluorophenethoxy)pyrimidine
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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A potential PET radiotracer for the 5-HT2C receptor: Synthesis and in vivo evaluation of
4-(3-[18F]fluorophenethoxy)pyrimidine
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Juhyeon Kim,1.2 Byung Seok Moon,3 Byung Chul Lee,3 HoꢀYoung Lee,3,* HakꢀJoong Kim,2
Hyunah Choo,1,4 Ae Nim Pae,4,5 Yong Seo Cho,1,4 SunꢀJoon Min,6,*
1Center for NeuroꢀMedicine, Korea Institute of Science and Technology (KIST), 5
Hwarangno 14ꢀgil, Seongbukꢀgu, Seoul, 02792, Republic of Korea
2Department of Chemistry, Korea University, Seoul, 02841, Republic of Korea
3Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam
13620, Republic of Korea
4Department of Biological Chemistry, Korea University of Science and Technology (UST),
217 Gajungro, Yuseongꢀgu, Daejeon, 34113, Republic of Korea
5Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, KIST,
Seoul 02792, Republic of Korea
6Department of Chemical & Molecular Engineering/Applied Chemistry, Hanyang University,
Ansan, Gyeonggiꢀdo, 15588, Republic of Korea.
*To whom correspondence should be addressed:
SunꢀJoon Min, Ph.D., Department of Chemical & Molecular Engineering/Applied Chemistry,
Hanyang University, 55 Hanyangdaehakꢀro, Sangnok, Ansan, Gyeonggiꢀdo 15588, Republic
of Korea (P) +82ꢀ31ꢀ400ꢀ5502/(F) +82ꢀ31ꢀ400ꢀ5457; Email: sjmin@hanyang.ac.kr
HoꢀYoung Lee, M.D., Department of Nuclear Medicine, Seoul National University Buandang
Hospital, 82 Gumiꢀro 173, Bundang, Seongnam, Gyeonggiꢀdo, 13620, Republic of Korea;
Email: debobkr@gmail.com
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Abstract: The serotonin 2C receptor subtype (5ꢀHT2C) is an excitatory 5ꢀHT receptor widely
distributed throughout the central nerve system. As the 5ꢀHT2C receptor displays multiple
actions on various neurotransmitter systems including glutamate, dopamine, epinephrine and
γꢀaminobutyric acid (GABA), abnormalities of the 5ꢀHT2C receptor are associated with
psychiatric diseases such as depression, schizophrenia, drug abuse, and anxiety. Up to date,
three kinds of 5ꢀHT2C PET radiotracers such as [11C]Nꢀmethylated arylazepine (1),
[11C]WAYꢀ163909 (2), [18F]fluorophenylcyclopropane (3) have been developed, but they may
not be suitable for in vivo 5ꢀHT2C imaging study due to their modest specific binding. Herein,
the synthesis and in vivo evaluation of 4ꢀ(3ꢀ[18F]fluorophenethoxy)pyrimidine [18F]4 as a
potential PET radiotracer for the 5ꢀHT2C 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/ꢁmol specific radioactivity. The in vivo PET imaging studies of [18F]4 with or without
locaserin, an FDA approved selective 5ꢀHT2C agonist, demonstrated that [18F]4 exhibits a
high level of specific binding to 5ꢀHT2C receptors in the rat brain.
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Keywords: 5ꢀHT2C receptor, in vivo, PET radioligands, brain imaging, psychiatric disorders
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GRAPHICAL TABLE OF CONTENTS
OTs
1) nBu4[
18F]F, TEAB,
I
Boc
18F
N
NH
TEMPO, 130 °C, 10 min
O
F
N
N
O
F
N
N
2) 2N HCl, 100 °C,
10 min
N
N
RCY = 7.8±2.7%
S.A.
= 89±14 GBq/ꢀmol
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With
blocking
Without
blocking
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Serotonin (5ꢀhydroxytryptamine, 5ꢀHT), 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.1ꢀ4 There are 14 distinct 5ꢀHT receptor subtypes, most of which belong
to a group of Gꢀprotein coupled receptors (GPCR). On the basis of their structural homology,
amino acid sequence, and signalling pathway, they are categorized into one of seven 5ꢀHT
classes (5ꢀHT1ꢀ7). The 5ꢀHT2C receptor is an excitatory 5ꢀHT receptor widely distributed
throughout the CNS. 5ꢀHT2C 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 5ꢀHT2C 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.6ꢀ8 As the 5ꢀHT2C receptor
displays multiple actions on various neurotransmitters and receptors, abnormalities of 5ꢀHT2C
receptors are associated with psychiatric diseases such as depression, schizophrenia, drug
abuse, and anxiety.9ꢀ15 Although a number of 5ꢀHT2C modulators have been developed as
chemical tools for the pharmacological study of the 5ꢀHT2C receptor,16ꢀ18 it is difficult to
elucidate the role of the 5ꢀHT2C receptor in diseases due to the lack of suitable methods for
determining the 5ꢀHT2C concentration in vivo.
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Figure 1. Examples of 5ꢀHT2C radioligands for PET molecular imaging
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Positron emission tomography (PET), one of the nonꢀinvasive 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 5ꢀHT
receptor subtypes to evaluate their function in physiology and psychiatric disorders, there are
no readily available radiotracers for in vivo imaging of the 5ꢀHT2C receptor.19ꢀ21 Recently,
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three 5ꢀHT2C PET tracers have been developed based on potent and selective
agonists
5ꢀHT2C
as shown in Figure 1.22ꢀ24 Compounds 1 and 2 were synthesized using efficient radiolabelling
processes with different carbonꢀ11 sources such as methyl iodide and formaldehyde, but their
preliminary PET imaging data in animal models represent nonꢀspecific binding. [18F]ꢀlabelled
compound 3, synthesized via metalꢀcatalysed arene radiofluorination, showed excellent brain
penetration and metabolic stability. However, it is not suitable for in vivo 5ꢀHT2C imaging
due to its modest specific binding. Thus, new 5ꢀHT2C radiotracers with improved specific
binding in vivo are required for understanding the full potential of the serotonergic system in
psychiatric disorders.
Among reported 5ꢀHT2C agonists,25ꢀ28 we selected pyrimidine analogue 4 as a potential target
molecule for 5ꢀHT2C 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 5ꢀHT subtypes. In particular, it is reported that 4 did not show any
significant agonistic effect on the 5ꢀHT2A receptor (EC50 of 5ꢀHT2C = 1.7 nM, Emax = 88% vs
EC50 of 5ꢀHT2A = 4441 nM, Emax = 62%),29 which is crucial for imaging a brain region of
high 5ꢀHT2C 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
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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).30ꢀ38
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In the current study, we report an efficient synthesis of [18F]ꢀlabelled pyrimidine 4 and in vivo
evaluation of this compound as a 5ꢀHT2C 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 5ꢀHT subtypes. The assay results showed that
4 has high binding affinities to 5ꢀHT2A, 5ꢀHT2B, 5ꢀHT2C, 5ꢀHT6 and 5ꢀHT7 with >90%
binding at a concentration of 10 ꢁM. (Table 1). Further determination of Ki values against 5ꢀ
HT receptor subtypes indicated that 4 displayed the highest binding affinity to 5ꢀHT2C as
anticipated. Most importantly, we observed at least 11ꢀfold selectivity for 5ꢀHT2C 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 5ꢀHT receptor subtypesa
5ꢀHT
subtypes
1A
1B
1D
78.3
542.0
1E
2A
2B
97.4
7.9
2C
98.2
0.7
3
5A
6
7
% binding
at 10 ꢁM
89.0
353.0
55.1
65.6
95.6
128.0
75.0
241.0
37.4
NDb
93.8
43.0
94.7
84.0
Ki (nM)
1780.0
1160.0
a5ꢀHT receptor binding was determined by competitive binding assay using [3H]ꢀlabelled compound as a radioligand for the
specific subtype (see Table S3). b Not determined
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Scheme 2. Synthesis of arylstannane 6. Reagents and conditions: (a) (
R
)ꢀ(+)ꢀpropylene oxide,
CuI, THF, rt, 78%; (b) 2,4ꢀdichloroꢀ5ꢀfluoropyrimidine, NaOtꢀBu, toluene, rt, 91%; (c) (R)ꢀ
(+)ꢀ1ꢀBocꢀ3ꢀmethylpiperazine, 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 2,4ꢀdichloroꢀ5ꢀfluoropyrimidine selectively afforded pyrimidine 10
in 91% yield, which was subjected to a second SNAr reaction with NꢀBocꢀpiperazine 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
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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
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9
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 electronꢀrich ring.
Thus, four different kinds of diaryliodonium salts 17a-d were synthesized with a modified
version of the literature procedure.40ꢀ43 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 sideꢀproduct 18 or
19. (Table S2). Although compounds 17b-d consist of the more electronꢀrich 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.
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OH
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OAc
I
a or b
c
Ar
I
OTs
Ar
Ar
OAc
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16a-d
15a (Ar = toluene)
15b (Ar = anisole)
15c (Ar = 2-thiophene)
15d (Ar = 3-thiophene)
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d
OTs
R
H
I
Boc
N
Ar
N
e,f
O
F
N
N
N
O
F
N
N
N
4 (R = F)
18 (R = I)
19 (R = H)
17a-d
Scheme 3. Synthesis and fluorination of diaryliodonium tosylate 17. Reagents and conditions:
(a) NaBO3, AcOH, 50 °C, 30ꢀ47%; (b) NaIO4, NaOAc, AcOH, Ac2O, 120 °C, 91%; (c)
TsOHꢀH2O, CH3CN, 0 °C; (d) 6, CHCl3, reflux (100−120 °C), 50ꢀ79%; (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 18Fꢀlabeled 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
(n n
Bu4N[18F]F) as shown in Scheme 4. Thus, a solution of 17a and Bu4N[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 Sepꢀ
pak 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/ꢁmol at
the end of the synthesis.
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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 5ꢀHT2C 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 5ꢀHT2C receptors, [18F]4
was coꢀadministrated with cold compound 4 or locaserin, an FDA approved selective 5ꢀHT2C
agonist (7.5ꢀfold higher binding affinity for 5ꢀHT2C over 5ꢀHT2A44) (Figure 2D and 2F).
Although the offꢀtarget 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 coꢀtreatment 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 5ꢀHT2C receptors.
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[18F]4
(B)
(A)
(C)
(E)
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(D)
[18F]4 + cold 4
[18F]4 + locaserin
(F)
Figure 2. PET analysis of [18F]4 in normal rats. (A) Summation image of [18F]4 dynamic
PET/CT data. (B) Timeꢀactivity curves for brain regions of interest in [18F]4 study. (C)
Summation image of [18F]4 dynamic PET/CT data with coꢀtreatment with cold compound 4.
(D) Timeꢀactivity curves for brain regions after coꢀtreatment with cold compound 4. (E)
Summation image of [18F]4 dynamic PET/CT data with coꢀtreatment with locaserin. (F)
Timeꢀactivity curves for brain regions after coꢀtreatment with locaserin. The PET images
shown were acquired using same rats.
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In conclusion, we successfully synthesized a 4ꢀ(3ꢀ[18F]fluorophenethoxy)pyrimidine as a
potential PET radiotracer for 5ꢀHT2C receptors via the SNAr reaction. Use of (4ꢀ
methylphenyl)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 5ꢀHT2C receptors.
Therefore, [18F]4 is a suitable candidate for in vivo 5ꢀHT2C PET imaging, which will be
further studied for clinical development.
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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
pꢀanisaldehyde stain followed
by brief heating on hot plate. Flash column chromatography was performed using silica gel
1
13
60 (230ꢀ400 mesh, Merck) with the indicated solvents. H and C 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 =
1
multiplet), integration, and coupling constant (
J
) in Hertz (Hz). H NMR chemical shifts are
13
reported relative to CDCl3 (7.26 ppm). C NMR was recorded relative to the central line of
CDCl3 (77.0 ppm). LC/MS analyses were performed on Shimadzu LCMSꢀ2020 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 tertꢀbutoxide (927 mg, 9.65
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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, 2,4ꢀdichloroꢀ5ꢀfluoropyrimidine (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
10
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17
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column chromatography on silica gel (EtOAc:
91%) as a colorless oil; Rf = 0.60 (EtOAc:
n
ꢀhexane = 1:8) to afford pyrimidine 10 (1.30 g,
1
n
ꢀhexane = 1:4). HꢀNMR (400 MHz, CDCl3)
δ
8.13 (d,
J
= 2.3 Hz, 1H), 7.21ꢀ7.18 (m, 1H), 6.83ꢀ6.82 (m, 2H), 6.78ꢀ6.75 (m, 1H), 5.57ꢀ5.48
= 6.2 Hz, 3H). 13CꢀNMR (100 MHz,
160.1, 159.5 (d, 2J = 11 Hz), 153.7 (d, 4J = 5 Hz), 146.5 (d, 1J = 252 Hz), 144.4 (d,
2J = 20 Hz), 138.9, 130.0, 122.3, 115.6, 112.7, 76.8, 55.6, 42.7, 19.7. HRMSꢀESI (m/z): [M +
(m, 1H), 3.79 (s, 3H), 3.08ꢀ2.85 (m, 2H), 1.42 (d,
J
CDCl3)
δ
Na]+ calcd for C14H14ClFN2NaO2: 319.0620; found: 319.0622. Optical rotation for (
R)ꢀ10:
D
[
α
]
23 = ꢀ18.6 (
c
= 0.234, CHCl3).
)-1-(3-methoxyphenyl)propan-2-yl)oxy)pyrimidin-2-yl)-
(R
)-tert-Butyl-4-(5-fluoro-4-(((
R
3-methylpiperazine-1-carboxylate (11). To a solution of pyrimidine 10 (631 mg, 2.13 mmol)
in DMSO (21.3 mL) was added ( )ꢀ(+)ꢀ1ꢀBocꢀ3ꢀmethyl piperazine (511 mg, 2.55 mmol) and
R
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/nꢀhexane = 1:4) to afford
methyl piperazine carboxylate 11 (513 mg, 52%) as a colorless oil; Rf = 0.33 (EtOAc/nꢀ
1
hexane = 1:4). HꢀNMR (400 MHz, CDCl3)
δ
7.91 (d,
J
= 2.9 Hz, 1H), 7.18ꢀ7.14 (m, 1H),
= 13.1 Hz, 1H), 4.11ꢀ3.87(m,
6.79ꢀ6.72 (m, 3H), 5.43ꢀ5.35 (m,1H), 4.68 (s, 1H) 4.24 (d,
J
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2H), 3.75 (s, 3H), 3.13ꢀ3.06 (m, 3H) 2.82ꢀ2.77 (m, 2H), 1.45 (s, 9H), 1.33 (d, J = 6.2 Hz, 3H),
13
1.11 (d,
J
= 6.7 Hz, 3H). CꢀNMR (100 MHz, CDCl3)
δ
160.5, 158.6 (d, 2J = 11 Hz), 157.8
8
9
(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,
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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. HRMSꢀESI (m/z):
[M + Na]+ calcd for C24H33ClFN4NaO4: 483.2378; found: 483.2378. Optical rotation for (
R)ꢀ
D
11: [
3-((
α
]
23 = 48.3 (
c
= 0.323, CHCl3).
)-2-methylpiperazin-1-yl)pyrimidin-4-yl)oxy)propyl)phenol (12)
R
)-2-((5-Fluoro-2-((
R
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).
1HꢀNMR (400 MHz, CDCl3)
δ 7.91 (d, J = 2.8 Hz, 1H), 7.09ꢀ7.05 (m, 1H), 6.73ꢀ6.64 (m, 3H),
5.48ꢀ5.40 (m, 1H), 4.81ꢀ4.79 (m, 1H), 4.37 (d,
3.22ꢀ3.03 (m, 4H), 2.87ꢀ2.74 (m, 2H) , 1.35 (d,
J
= 12.0 Hz, 1H), 3.30 (d,
J = 12.3 Hz, 1H),
J
= 6.2 Hz, 3H), 1.30 (d, J
= 6.9 Hz, 3H). 13Cꢀ
NMR (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. HRMSꢀESI (m/z): [M + H]+ calcd for C18H24FN4O2: 347.1878; found:
D
347.1880. Optical rotation for (
R)ꢀ12: [
α]
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
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(8.23 mg, 0.0980 mmol) and diꢀtꢀbutyldicarbonate (22.0 mg, 0.0980 mmol). The reaction
6
7
8
9
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).
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1HꢀNMR (400 MHz, CDCl3) δ 7.94 (d,
5.44ꢀ5.35 (m, 1H), 4.70 (s, 1H), 4.26 (d,
3H), 2.93ꢀ2.78 (m, 2H), 1.49 (s, 9H), 1.36 (d,
J = 3.0 Hz, 1H), 7.16ꢀ7.12 (m, 1H), 6.79ꢀ6.68 (m, 3H),
J
= 13.4 Hz, 1H), 4.13ꢀ3.87 (m, 2H), 3.17ꢀ3.06 (m,
= 6.7 Hz, 3H). 13Cꢀ
J
= 6.2 Hz, 3H), 1.15 (d, J
NMR (100 MHz, CDCl3) δ 158.6 (d, 2J = 3 Hz), 157.4 (d, 4J = 3 Hz), 156.8, 156.1, 143.6 (d,
2J = 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. HRMSꢀESI (m/z): [M + Na]+ calcd for C23H31FN4NaO4:
D
469.2222; found: 469.2220. Optical rotation for (
)-tert-Butyl-4-(5-fluoro-4-(((
R)ꢀ13: [
α]
24 = 97.9 (
c
= 0.286, CHCl3).
(R
R
)-1-(3-(((trifluoromethyl)sulfonyl)oxy) phenyl)propan-2-
yl)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,Nꢀdiisopropylethylamine
(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:
nꢀhexane = 1:4)
to afford triflate 14 (64.0 mg, 91%) as a colorless oil; Rf = 0.41 (EtOAc:
n
ꢀhexane = 1:4). 1Hꢀ
NMR (400 MHz, CDCl3)
δ
7.95 (d,
J
= 2.9 Hz, 1H), 7.38ꢀ7.34 (m, 1H), 7.27ꢀ7.25 (m, 2H),
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7.17ꢀ7.12 (m, 1H), 5.60ꢀ5.38 (m, 1H), 4.76ꢀ4.68 (m, 1H), 4.27ꢀ3.91 (m, 3H), 3.17ꢀ3.09 (m,
3H), 3.01ꢀ2.87 (m, 2H), 1.48 (s, 9H), 1.37 (d,
157.4 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 149.6, 143.4 (d,
2J = 20 Hz), 140.6, 139.9 (d, 1J = 247 Hz), 130.1, 129.6, 122.2, 119.4, 118.7 (q,
J = 6.2 Hz, 3H), 1.13 (d, J
= 6.7 Hz, 3H). 13Cꢀ
NMR (100 MHz, CDCl3)
δ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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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. HRMSꢀESI (m/z): [M + Na]+
D
calcd for C24H30F4N4NaO6S: 601.1714 ; found: 601.1713. Optical rotation for (
= ꢀ6.1 ( = 0.245, CHCl3).
)-tert-Butyl-4-(5-fluoro-4-(((R)-1-(3-(tributylstannyl)phenyl)propan-2-yl)oxy)
R
)ꢀ14: [
α]
24
c
(R
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:nꢀ
hexane = 1:8) to afford arylstannane 6 (662 mg, 63%) as a colorless oil; Rf = 0.71 (EtOAc:nꢀ
1
hexane = 1:4). HꢀNMR (400 MHz, CDCl3)
δ
7.93 (d,
J
= 2.9 Hz, 1H), 7.31ꢀ7.22 (m,
3H),
7.15 (d,
J
= 7.6 Hz, 1H), 5.44ꢀ5.36 (m, 1H), 4.69 (s, 1H), 4.27ꢀ4.24 (d,
J
= 2.9 Hz , 1H), 4.14ꢀ
2.89 (m, 2H), 3.15ꢀ3.08 (m, 3H), 2.86ꢀ2.81 (m, 2H), 1.53ꢀ1.49 (m, 6H), 1.47 (s, 9H), 1.36ꢀ
= 7.3 Hz, 9H). 13Cꢀ
157.7 (d, 2J = 11 Hz), 156.8 (d, 4J = 2 Hz), 155.2, 143.1 (d, 2J
=
1.28 (m, 9H), 1.13 (d,
J = 6.7 Hz, 3H) 1.02 (t, J =8.1 Hz, 6H), 0.86 (t, J
NMR (100 MHz, CDCl3)
δ
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. HRMSꢀESI (m/z): [M +
Na]+ calcd for C35H57FN4NaO3Sn: 743.3336 ; found: 743.3334. Optical rotation for (
R)ꢀ6:
D
[
α]
24 = ꢀ6.5 (
c
= 0.123, CHCl3).
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(3-((R
)-2-((2-((
R
)-4-(tert-Butoxycarbonyl)-2-methylpiperazin-1-yl)-5-fluoropyrimidin-4-
6
7
yl)oxy) propyl)phenyl)(
p-tolyl)iodonium 4-methylbenzene sulfonate (17a). To a solution
8
9
of diacetate 15a46 (9.30 mg, 0.0490 mmol) in CH3CN (0.250 mL) was added
p
ꢀTsOHꢀH2O
10
11
12
13
14
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17
18
19
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23
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(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
1
(DCM/MeOH = 10:1). HꢀNMR (400 MHz, CDCl3)
δ
7.93 (d,
= 8.2 Hz, 1H), 7.32 (t, = 7.9 Hz, 1H),
= 8.1 Hz, 2H), 5.36ꢀ5.31 (m, 1H), 4.66 (d, J = 3.9 Hz, 1H),
J = 2.8Hz, 1H), 7.83 (s, 1H),
7.78ꢀ7.74 (m,
3
H), 7.66 (d,
J
= 8.1 Hz, 2H), 7.44 (d,
J
J
7.19 (d, = 8.4 Hz, 2H), 7.09 (d,
J
J
4.26ꢀ3.87 (m, 2H), 3.14ꢀ2.93 (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). 13CꢀNMR (100 MHz, CDCl3) 157.3 (d, 2J = 11 Hz),
δ
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. HRMSꢀESI (m/z): [M + Na]+
D
calcd for C30H37FIN4O3: 647.1889 ; found: 647.1877. Optical rotation for (
R)ꢀ17a: [
α]
=
24
164.1 ( = 0.156, CHCl3).
c
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 (TrisꢀHCl), 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 ꢁM, 5 ꢁM, and 50 ꢁM. The radioligand (Table S3)
was diluted to five times the assay concentration in standard binding buffer. Aliquots (50 mL)
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of the radioligand were dispensed into the wells of a 96ꢀwell 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 96ꢀwell Brandel harvester.
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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, 4ꢀml 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® PSꢀHCO3 cartridge and eluted with methanol/water (1 mL/0.1 mL) containing
tetrabutylammonium bicarbonate (40 wt%, 2 ꢀ
L).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 SepꢀPak 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
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tC18 SepꢀPak 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
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M) at a flow rate of 3.0 mL/min, using a UV detector at 254 nm and a γꢀray 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% ethanolꢀsaline solution by tC18 SepꢀPak cartridge to remove the clinically
unavailable HPLC solvent. The isolated radiochemical yield was 7.8 ± 2.7 % (n = 6, decay
corrected) and produced approximately 1.7ꢀ2.2 GBq per batch. An aliquot of the formulated
solution was checked by analytical HPLC (Waters, Xterra RPꢀ18, 5 ꢁm, 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/ꢁmol 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, Xꢀray 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 TearꢀTomo Real 3D PET engine (nanoScan PET/CT, Mediso Inc.,
Budapest, Hugary). To evaluate specific binding to 5ꢀHT2C receptor, 10 mg/kg of locaserin or
cold form 4 was coincidentally injected with 1.0 mCi of [18F]4. Images were acquired right
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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.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: sjmin@hanyang.ac.kr. Fax: 82ꢀ31ꢀ400ꢀ5457. Tel: 82ꢀ31ꢀ400ꢀ5502.
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 (NRFꢀ2016R1A2B1012277 and NRFꢀ
2014M3C1A3054141).
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 (HHSNꢀ271ꢀ2008ꢀ00025ꢀC). Animal
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experimentation was performed according to Guidelines for Accommodation and Care of
Animals.
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REFERENCES
1. Barnes, N. M., and Sharp, T. A. (1999) A review of central 5ꢀHT receptors and their
function. Neuropharmacology 38, 1083–1152.
2. Monti, J. M. (2010) The role of dorsal raphe nucleus serotonergic and nonꢀserotonergic
neurons, and of their receptors, in regulating waking and rapid eye movement (REM) sleep.
Sleep Med. Rev. 14, 319–327.
3. Schmitt, J. A., Wingen, M., Ramaekers, J. G., Evers, E. A., and Riedel, W. J. (2006)
Serotonin and human cognitive performance. Curr. Pharm. Des. 12, 2473–2486.
4. Chagraoui, A., Thibaut, F., Skiba, M., Thuillez, C., and Bourin, M. (2016) 5ꢀHT2C
receptors in psychiatric disorders: A review. Prog. NeuroꢀPsychoph. 66, 120–135.
5. Clemett, D. A., Punhani, T., Duxon, M. S., Blackburn, T. P., and Fone, K. C. (2000)
Immunohistochemical localisation of the 5ꢀHT2C receptor protein in the rat CNS.
Neuropharmacology 39, 123–132.
6. Hoyer, D., Engel, G., and Kalkman, H. O. (1985) Molecular pharmacology of 5ꢀHT1 and
5ꢀHT2 recognition sites in rat and pig brain membranes: radioligand binding studies with
[3H]5ꢀHT, [3H]8ꢀOHꢀDPAT, (–)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin.
Eur. J. Pharmacol. 118, 13–23.
7. Hoyer, D., Pazos, A., Probst, A., and Palacios, J. M. (1986) Serotonin receptors in the
human brain. II. Characterization and autoradiographic localization of 5ꢀHT1C and 5ꢀHT2
recognition sites. Brain Research 376, 97–107.
8. Pazos, A., Hoyer, D., and Palacios, J. M. (1984) Mesulergine, a selective serotoninꢀ2
ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence
20
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ACS Chemical Neuroscience
Page 22 of 26
1
2
3
4
5
6
7
8
9
for species differences in brain serotoninꢀ2 receptors. Eur. J. Pharmacol. 106, 531–538.
9. Higgins, G. A., and Fletcher, P. J. (2003) Serotonin and drug reward: focus on 5ꢀHT2C
receptors. Eur. J. Pharmacol. 480, 151–162.
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
10. Giorgetti, M., and Tecott, L. H. (2004) Contributions of 5ꢀHT2C receptors to multiple
actions of central serotonin systems. Eur. J. Pharmacol. 488, 1–9.
11. Millan, M. J. (2005) Serotonin 5ꢀHT2C receptors as a target for the treatment of depressive
and anxious states: focus on novel therapeutic strategies. Thérapie 60, 441–460.
12. RosenzweigꢀLipson, S., Comery, T. A., Marquis, K. L., Gross, J., and Dunlop, J. (2012)
5ꢀHT2C agonists as therapeutics for the treatment of schizophrenia. Handb. Exp. Pharmacol
213, 147–165.
.
13. De Deurwaerdere, P., Lagiere, M., Bosc, M., and Navailles, S. (2013) Multiple controls
exerted by 5ꢀHT2C receptors upon basal ganglia function: from physiology to
pathophysiology. Exp. Brain Res. 230, 477–511.
14. Higgins, G. A., and Fletcher, P. J. (2015) Therapeutic potential of 5ꢀHT2C receptor
agonists for addictive disorders. ACS Chem. Neurosci. 6, 1071–1088.
15. Howell L. L., and Cunningham K. A. (2015) Serotonin 5ꢀHT2 receptor interactions with
dopamine function: implications for therapeutics in cocaine use disorder. Pharmacol. Rev. 67
,
176–197.
16. Lee, J., Jung, M. E., and Lee, J. (2010) 5ꢀHT2C receptor modulators: a patent survey.
Expert Opin. Ther. Pat. 20, 1429–1455.
17. Monck, N. J. T., and Kennett, G. A. (2008) 5ꢀHT2C ligands: recent progress. Prog. Med.
Chem. 46, 281–390.
18. Sargent, B. J., and Henderson, A. J. (2011) Targeting 5ꢀHT receptors for the treatment of
obesity. Curr. Opin. Pharmacol. 11, 52–58.
19. Paterson, L. M., Tyacke, R. J., Nutt, D. J., and Knudsen, G. M. (2010) Measuring
21
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Page 23 of 26
ACS Chemical Neuroscience
1
2
3
4
5
6
7
endogenous 5ꢀHT release by emission tomography: promises and pitfalls. J. Cereb. Blood F.
Met. 30, 1682–1706.
8
9
20. Zimmer, L., and Le Bars, D. (2013) Current status of positron emission tomography
radiotracers for serotonin receptors in humans. J. Label Compd. Radiopharm. 56, 105–113.
21. Paterson, L. M., Kornum, B. R., Nutt, D. J., Pike, V. W., and Knudsen, G. M. (2013) 5ꢀ
HT radioligands for human brain imaging with PET and SPECT. Med. Res. Rev. 33, 54–111.
22. Granda, M. L., Carlin, S. M., Moseley, C. K., Neelamegam, R., Mandeville, J. B., and
Hooker, J. M. (2013) Synthesis and Evaluation of Methylated Arylazepine Compounds for
PET Imaging of 5ꢀHT2C Receptors. ACS Chem. Neurosci. 4, 261–265.
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
23. Neelamegam, R., Hellenbrand, T., Schroeder, F. A., Wang, C., Hooker, J. M. (2014)
Imaging evaluation of 5HT2C agonists, [11C]WAYꢀ163909 and [11C]Vabicaserin, formed by
PictetꢀSpengler cyclization. J. Med. Chem. 57, 1488–1494.
24. Kamlet, A. S., Neumann, C. N., Lee, E., Carlin, S. M., Moseley, C. K., Stephenson, N.,
Hooker J. M., and Ritter, T. (2013) Application of palladiumꢀmediated 18Fꢀfluorination to
PET radiotracer development: Overcoming hurdles to translation. PlosOne 8, e59187.
25. Green, M. P., McMurray, G., and Storer, R. I. (2016) Selective 5ꢀHT2C receptor agonists:
Design and synthesis of pyridazineꢀfused azepines. Bioorg. Med. Chem. Lett. 26, 4117–4121.
26. Cheng, J., Giguere, P. M., Schmerberg, C. M., Pogorelov, V. M., Rodriguiz, R. M., Huang,
X.ꢀP., Zhu, H., McCorvy, J. D., Wetsel, W. C., Roth, B. L., and Kozikowski, A. P. (2016)
Further advances in optimizing (2ꢀphenylcyclopropyl)methylamines as novel serotonin 2C
agonists: Effects on hyperlocomotion, prepulse inhibition, and cognition models. J. Med.
Chem. 59, 578–591.
27. Shidore, M., Machhi, J., Murumkar, P., Barmade, M., Thanki, J., and Yadav, M. R. (2015)
3ꢀSubstituted 1ꢀmethylꢀ3ꢀbenzazepinꢀ2ꢀones as 5ꢀHT2C receptor agonists. RSC Adv. 5
,
91908–91921.
22
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1
2
3
4
5
6
7
8
9
28. Cheng, J., Giguère, P. M., Onajole, O. K., Lv, W., Gaisin, A., Gunosewoyo, H.,
Schmerberg, C. M., Pogorelov, V. M., Rodriguiz, R. M., Vistoli, G., Wetsel, W. C., Roth, B.
L., Kozikowski, A. P. (2015) Optimization of 2ꢀphenylcyclopropylmethylamines as selective
serotonin 2C receptor agonists and their evaluation as potential antipsychotic agents. J. Med.
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
Chem. 58, 1992–2002.
29. Kalgutkar, A. S., Bauman, J. N., McClure, K. F., Aubrecht, J., Cortina, S. R., and Paralkar,
J. (2009) Biochemical basis for differences in metabolismꢀdependent genotoxicity by two
diazinylpiperazineꢀbased 5ꢀHT2C receptor agonists. Bioorg. Med. Chem. Lett. 19, 1559–1563.
30. Pike, V. W., and Aigbirhio, F. I., (1995) Reactions of cyclotronꢀproduced [18F]fluoride
with diaryliodonium salts ꢀ a novel singleꢀstep route to noꢀcarrierꢀadded [18 F]fluoroarenes. J.
Chem. Soc., Chem. Commun. 2215–2216.
31. Ross, T. L., Ermert, J., Hocke, C., and Coenen, H. H. (2007) Nucleophilic 18Fꢀfluorination
of heteroaromatic iodonium salts with noꢀcarrierꢀadded [18F]fluoride. J. Am. Chem. Soc
. 129,
8018–8025.
32. Cai, L., Lu, S., and Pike, V. W. (2008) Chemistry with [18F]fluoride ion. Eur. J. Org.
Chem. 2853–2873.
33. Preshlock, S., Tredwell, M., and Gouverneur, V. (2016) 18FꢀLabeling of arenes and
heteroarenes for applications in positron emission tomography. Chem. Rev. 116, 719–766.
34. Rotstein, B. H., Stephenson, N., Vasdev, N., and Liang, S. H. (2014) Spirocyclic
hypervalent iodine(III)ꢀmediated radiofluorination of nonꢀactivated and hindered aromatics”
Nat. Comm. 5, 4365–4369.
35. Shah, A., Pike, V. W., and Widdowson, D. A. (1998) The synthesis of [18F]fluoroarenes
from the reaction of cyclotronꢀproduced [18F]fluoride ion with diaryliodonium salts. J. Chem.
Soc. Perkin Trans. 1 2043–2046.
36. Yusubov, M. S., Maskaev, A. V., and Zhdankin, V. V. (2011) Iodonium salts in organic
23
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Page 25 of 26
ACS Chemical Neuroscience
1
2
3
4
5
synthesis. ARKIVOC 370–409.
6
7
8
9
37. Yusubov, M. S., Svitich, D. Y., Larkina, M. S., and Zhdankin, V. V. (2013) Applications
of iodonium salts and iodonium ylides as precursors for nucleophilic fluorination in positron
emission tomography. ARKIVOC 364–395.
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
38. Carroll, M. A., Jones, C., and Tang, S.ꢀL. (2007) Fluoridation of 2ꢀthienyliodonium salts.
J. Labelled Compd. Radiopharm. 50, 450–451.
39. Gan, T., Van Ornum, S. G., and Cook, J. M. (1997) The synthesis of GABAA active
ligands by the Stille process. Tetrahedron Lett. 38, 8453–8456.
40. Merritt, E. A., Carneiro, V. M. T., Silva L. F., Jr., and Olofsson, B. (2010) Facile synthesis
of Koser's reagent and derivatives from iodine or aryl iodides. J. Org. Chem. 75, 7416–7419.
41. Nabana, T., and Togo, H. (2002) Reactivities of novel [hydroxy(tosyloxy)iodo]arenes and
[hydroxy(phosphoryloxy)iodo]arenes for αꢀtosyloxylation and αꢀphosphoryloxylation of
ketones. J. Org. Chem. 67, 4362–4365.
42. Zhang, M. R., Kumata, K., and Suzuki, K. (2007) A practical route for synthesizing a
PET ligand containing [18F]fluorobenzene using reaction of diphenyliodonium salt with [18F]
Fꢀ. Tetrahedron Lett. 48, 8632–8635.
43. Moon, B. S., Kil, H. S., Park, J. H., Kim, J. S., Park, J., Chi, D. Y., Lee, B. C., and Kim, S.
E. (2011) Facile aromatic radiofluorination of [18F]flumazenil from diaryliodonium salts with
evaluation of their stability and selectivity. Org. Biomol. Chem. 9, 8346–8355.
44. Thomsen, W. J., Grottick, A. J., Menzaghi, F., ReyesꢀSaldana, H., Espitia, S., Yuskin, D.,
Whelan, K., Martin, M., Morgan, M., Chen, W., AlꢀShamma, H., Smith, B., Chalmers, D.,
and Behan, D. (2008) Lorcaserin, a novel selective human 5ꢀhydroxytryptamine 2C agonist:
in vitro and in vivo pharmacological characterization. J. Pharmacol. Exp. Ther. 325, 577–587.
45. Serafine, K. M., Rice, K. C., and France, C. P. (2015) Directly observable behavioral
effects of lorcaserin in rats. J. Pharmacol. Exp. Ther. 355, 381–385.
24
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9
46. Hossain, M. D. and Kitamura, T. (2005) Alternative, easy preparation of
(diacetoxyiodo)arenes from iodoarenes using potassium peroxodisulfate as the Oxidant.
Synthesis 1932–1934.
10
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60
47. Moon, B. S., Kim, B. S., Park, C. Jung, J. H. Lee, Y. W. Lee, H.–Y., Chi, D. Y., Lee, B. C.,
and Kim, S. E. (2014) [18F]FluoromethylꢀPBR28 as a potential radiotracer for TSPO:
preclinical comparison with [11C]PBR28 in a rat model of neuroinflammation. Bioconjugate
Chem. 25, 442–450.
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