Synthesis and Evaluation of a New 18F-Labeled Radiotracer for Studying the GABAB Receptor in the Mouse Brain

Article abstract of DOI:10.1021/acschemneuro.8b00038

New GABA_B agonists, fluoropyridyl ether analogues of baclofen, have been synthesized as potential PET radiotracers. The compound with highest inhibition binding affinity as well as greatest agonist response, (R)-4-amino-3-(4-chloro-3-((2-fluoro

Full text of DOI:10.1021/acschemneuro.8b00038

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Article
18
Synthesis and evaluation of a new F-labeled radiotracer
B
for studying the GABA receptor in the mouse brain
Ravi Naik, Heather Valentine, Robert F. Dannals, Dean F Wong, and Andrew G. Horti
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00038 • Publication Date (Web): 02 Mar 2018
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Synthesis and evaluation of a new 18F-labeled radiotracer for studying the
GABAB receptor in the mouse brain
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Ravi Naik†, Heather Valentine†, Robert F. Dannals†, Dean F. Wong†, Andrew G. Horti†*
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†Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, 21287
USA.
ABSTRACT:
New GABAB agonists, fluoropyridyl ether analogues of baclofen, have been synthesized as potential PET
radiotracers. The compound with highest inhibition binding affinity as well as greatest agonist response,
(R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀfluoropyridinꢀ4ꢀyl)methoxy)phenyl)butanoic acid (1b), was radiolabeled
with 18F with good radiochemical yield, high radiochemical purity, and high molar radioactivity. The
regional brain distribution of the radiolabeled (R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀ[18F]fluoropyridinꢀ4ꢀ
yl)methoxy)phenyl)butanoic acid, [18F]1b, was studied in CDꢀ1 male mice. The study demonstrated that
[18F]1b enters the mouse brain (1% ID/g tissue). The accumulation of [18F]1b in the mouse brain was
inhibited (35%) by preꢀinjection of GABAB agonist 1a suggesting that the radiotracer brain uptake is
partially mediated by GABAB receptors. The presented data demonstrated a feasibility of imaging of
GABAB receptors in rodents and justify further development of GABAB PET tracers with improved
specific binding and greater bloodꢀbrain barrier permeability.
Keywords: GABAB receptor, PET, radiotracer, autism, bloodꢀbrain barrier permeability
INTRODUCTION
GABAB is a receptor subclass for the neurotransmitter GABA (4ꢀaminobutanoic acid).1 Recent
reviews summarize evidence that GABAB receptors play an essential role in various central and
peripheral disorders.2, 3 Molecules that modulate GABAB receptors are of great medicinal interest for the
treatment of many disorders and conditions including alcohol dependence, antiꢀnociception, spasticity,
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fragile X syndrome, Austin’s disease, retinal ganglion cell degeneration, and autism.4–9 The GABAB
agonist baclofen 1 is widely used in the treatment of gastroesophageal reflux, cancer pain, and overactive
bladder. Baclofen is under development for the treatment of behavioral symptoms of Fragile X
Disorder.4,10
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Autism is a neurodevelopmental disorder characterized by impairments in social interaction,
restricted and repetitive behavior, and deficits in language and communication.11 In 2006, in the USA and
Europe a prevalence rate of autism of 116 per 10,000 children has been reported.12
Existing therapeutic agents are not effective for treatment of core symptoms of autism spectrum
disorders.13 Recent preꢀclinical studies demonstrated that GABAB receptor agonist reverses social deficits
and reduces repetitive behavior in animal models of autism.14 Postꢀmortem studies have demonstrated that
GABAB receptors are reduced (42ꢀ79%) in the various brain regions of subjects suffering from autism.15
Many GABAB drugs are analogues of GABA. These analogues are polar compounds that exhibit
moderate BBB permeability. The recently developed baclofen derivative 1a (Fig. 1) demonstrated an
improved BBB permeability and exhibited at least an order of magnitude higher GABAB receptor potency
as compared to an active enantiomer of baclofen, Rꢀ(ꢀ)ꢀbaclofen (Rꢀ1) (pEC50 = 8.5 and 7.1,
respectively).4
Figure 1. Structure of Rꢀ(ꢀ)ꢀbaclofen (Rꢀ1) and potent brainꢀpenetrating GABAB agonist 1a4.
In vivo imaging and quantification of GABAB binding in the human brain would provide a significant
advance in the understanding of autism and other GABABꢀrelated CNS disorders and could also facilitate
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novel GABAB drug development. Positron emission tomography (PET) is the advanced technique to
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quantify neuronal receptors and their occupancy in vivo. Development of a suitable PET radiotracer for
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GABAB would be of substantial interest. Previously synthesized PET radiotracers 2-4
(Fig. 2) for
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imaging GABAB have shown negligible permeability of the bloodꢀbrain barrier (BBB) in animal
experiments.
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Figure 2. Previously reported GABAB PET radiotracers.
In this report, we describe the design, synthesis, 18Fꢀradiolabeling, and in vitro and in vivo
characterization in mice of fluoro derivatives of 1a as potential probes for PET imaging of GABAB
receptors.
RESULTS AND DISCUSSION
Chemistry:
Compound 1a was initially prepared as described elsewhere (Scheme 1)4. However, the conversion of
the intermediate iodo compound 9 to phenol 10 under the published conditions4 afforded very low yield
that is a common problem with the Suzuki reaction when an orthoꢀsubstituent is present.19 Therefore, we
designed a different methodology for the synthesis of the desired compound 10 (Schemes 2 and 3).
This route started with protection of 4ꢀchloroꢀ3ꢀhydroxybenzaldehyde 13 by using benzyl bromide in
the presence of an inorganic base and DMF. The Henry reaction of a 3ꢀ(benzyloxy)ꢀ4ꢀ
chlorobenzaldehyde 14 with nitromethane was used to give the nitrovinyl derivative 15 in good yields.
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Next, a Michael reaction of compound 15 using (R)ꢀ3ꢀacetylꢀ4ꢀbenzyloxazolidinꢀ2ꢀone 12 in the presence
of NaHDMS gave the intermediate 16.
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Reduction of the nitro group of compound 16 followed by cyclization of the resulting amine in situ
yielded 17 over two steps.20, 21 Protection of lactam 17 with tꢀBOC gave the NꢀBOC derivative 18.
Debenzylation of 18 with palladium hydroxide gave the desired phenol 10 as a key intermediate. The
intermediate 12 was synthesized from commercially available (R)ꢀ4ꢀbenzyloxazolidinꢀ2ꢀone 11 by using
nꢀBuLi and acetyl chloride in THF (Scheme 2).
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The phenol 10 reacted with pyridyl alcohol via a modified Mitsunobu22 reaction to give ether
derivatives 19a-c in high yields. The protected lactam 19a-c underwent acidic hydrolysis to give the
desired amino acid derivatives 1b-d as shown in Scheme 3.
Scheme 1. Synthesis of compound 1a.
Reagents and conditions: (a) Acetic acid, reflux, 12h, 99%; (b) Guanidine nitrate, H2SO4, 0 °C to rt, 1h,
95%; (c) Zn, NH4Cl, THF/MeOH/H2O, reflux, 1h, 97%; (d) NaNO2, KI, 12 N HCl, 0 °C to rt, 2.5 h, over
three steps, 82%; (e) Boc anhydride, K2CO3, MC, rt, 12h, 92%; (f) (i) PdCl2(dppf)2, octamethyl 2,2ꢀbisꢀ
1,3,2ꢀdioxaborolane, KOAc, DMSO, 120 °C, 12h; (ii) 35% H2O2, DCM, 12h, over two steps, 17%; (g) i)
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4ꢀ(bromomethyl)pyridine hydrobromide, K2CO3, Ethanol, 100 °C to rt, 12h, ii) 6N HCl, 90 °C to rt, 3h,
over two steps, 69%.
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Scheme 2. Synthesis of (R)ꢀ3ꢀacetylꢀ4ꢀbenzyloxazolidinꢀ2ꢀone.
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Reagents and conditions: (a) nꢀBuLi, Acetyl chloride, THF, ꢀ78 °C to rt, 4h, 89%.
Scheme 3. Synthesis of compounds 1b-d.
Reagents and conditions: a) Benzyl bromide, K2CO3, DMF, rt, 8h, 94%; b) Nitromethane, Ammonium
.
acetate, Acetic acid, 105 °C to rt, 1.5h, 86%; c) 14, NaHDMS, THF, ꢀ78 °C to rt, 2h, 53%; d) NiCl2 H2O,
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NaBH4, Ethanol, 0 °C to rt, 4h, 52%; e) Boc anhydride, DMAP, Et3N, MC, 0 °C to rt, 8h, 90%; f) Pd/C,
MeOH, rt, 0.5h, 80%; g) DIAD, PPh3, THF, 0 °C to rt, 15h, 77%ꢀ84%; h) 1,4ꢀDioxane HCl at 90 °C for
1b and 6N HCl at 90 °C for 1c and concentrated HBr at 90 °C for 1d, 12h, 37% to 73%.
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In vitro assay
The in vitro assay of compounds 1a-1c and the reference compound, Rꢀbaclofen (Rꢀ1) was performed
commercially (EurofinsꢀCEREP).
Functional agonist assay. The in vitro agonist activity assay at the human GABAB receptor expressed
in transfected CHO cells23 was performed by measuring an electric impedance modulation using the
Cellular Dielectric Spectroscopy (CDS) detection method.24
Substitution of the aromatic ring of Rꢀbaclofen (Rꢀ1) with 4ꢀpyridyl (1a)4 or fluoro 4ꢀpyridyl ether
(1b-1c) resulted in a striking increase in the GABAꢀB % agonist response (Fig. 3). The fluoro derivative
1b exhibited the highest GABAB agonist response within the series 1a-1c.
In vitro binding assay. In the GABAB radioligand binding assay studies, compound 1b exhibited a
greater binding inhibition than that of 1c (34% and 15%, respectively, at concentration of 100 nM).
Therefore, 1b has been selected for further radiolabeling and animal experiments.
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R-1 1a 1b 1c
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1.0E-8M
1.0E-7M
1.0E-6M
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Test concentration
Figure 3. GABAB agonist effect of compounds 1a-1c and Rꢀbaclofen (Rꢀ1) obtained by measuring an
electric impedance modulation in the human GABAB receptor transfected CHO cells using the Cellular
Dielectric Spectroscopy (CDS) detection method.
aValues are the means of % of control agonist response of two independent experiments, each in duplicate
± SD. Compound 1a was not tested at the concentration of 10ꢀ6 M.
Radiochemistry
The radiolabeling of 2ꢀfluoroꢀpyridine derivatives has been well established since the development of
18Fꢀderivatives of epibatidine.25, 26 The labeling of 1b with 18F (code name [18F]JHU11631) (Scheme 4)
was performed remotely in one step using a Kryptofix 222®, ꢀ assisted radiofluorination of the respective
bromoꢀprecursor 1d in a modified MicroLab radiochemistry box (GE). The radiolabeling required high
temperature (180oC) and DMSO was used as the reaction solvent. The synthesis was performed using
mild base, K2C2O4, under the conditions described previously.27 The reaction was followed by a semiꢀ
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preparative HPLC separation using the general procedure.25 The radiolabeled product [18F]1b and
precursor 1d were fully separated by preparative HPLC (Rt = 15.5 and 23 min, respectively). Precursor
was not detected by analytical HPLC in the final product solution.
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The final product [18F]1b was prepared with a radiochemical yield of 12ꢀ18% (n = 5, end of synthesis,
nonꢀdecayꢀcorrected), with a molar radioactivity in the range of 330–515 GBq/µmol (9–14 Ci/µmol) and
a radiochemical purity greater than 97%. The final product [18F]1b (72 ꢀ 86 mCi) was formulated as a
solution in sterile saline containing 7% ethanol.
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It is noteworthy that radiolabelling of [18F]1b did not require a protection of liable ꢀNH2 and ꢀCOOH
protons in the precursor molecule. This may be explained by a relatively large molecular fragment
between the 18Fꢀatom and liable protons and in agreement with 18Fꢀlabeling of other compounds with
liable protons.25
Scheme 4. Radiosynthesis of [18F]1b.
Biodistribution studies of [18F]1b in mice
Baseline studies in CD-1 mice.
The regional brain distribution of the radiotracer [18F]1b was studied in CDꢀ1 male mice. The
radioligand entered the mouse brain with peak (15 min) radioactivity accumulation of ~25%ID/g*body
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weight (~ 1%ID/g tissue) followed by rapid washout (Table 1). The observed brain uptake is moderate
and it is equal to the threshold uptake for selection of good PET tracer in our lab. The accumulation of
radioactivity was seen in various subcortical and cortical regions including superior colliculus, striatum,
hypothalamus, thalamus, cortex and cerebellum.
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Previous in vitro autoradiography studies demonstrated that GABAB is widely distributed throughout
the rodent brain regions.28ꢀ30 The published in vitro data are not sufficient for defining a detailed regional
brain distribution pattern, but the presence of GABAB was observed in the superior colliculus, thalamus
and molecular layers of cerebellum.28 The previous in vitro data agree with in vivo distribution of [18F]1b
in the CDꢀ1 mouse brain (Table 1).
Table 1. Regional brain uptake of [18F]1b at different timeꢀpoints post iv injection. Abbreviators: CB–
cerebellum; Hyp–hypothalamus; F. Ctx–frontal cortex; Ctx–cortex; Th–thalamus; Str–striatum; S. Coll–
superior colliculus; REST–the rest of brain. Data: mean %ID/g tissue*body weight ± SD (n = 6).
Regions
Hyp
15 min
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24.66
20.95
20.64
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22.54
21.51
23.45
SD
60 min
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4.23
3.55
3.16
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4.41
4.62
3.77
SD
120 min
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SD
240 min
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SD
1.65
3.29
2.83
3.25
2.96
3.68
2.92
3.13
1.71
0.65
0.59
0.53
0.46
0.59
0.84
0.42
1.02
0.58
0.37
0.29
0.47
0.34
0.28
0.39
0.62
0.48
0.25
0.22
0.33
0.29
0.19
0.18
2.31
1.74
Str
1.53
1.10
F.Ctx
Ctx
1.27
0.88
1.90
1.43
Th
1.92
1.43
S.Coll
CB
1.61
1.09
1.58
0.94
REST
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Specificity of [18F]1b Binding in the Mouse Brain
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A conventional in vivo blockade methodology was used for demonstration of the binding specificity of
[18F]1b radiotracer at the GABAB receptor in the mouse brain. A blockade study with a drug that is highly
selective at the target binding site is expected to show the specificity of the radioligand binding. In the
blocking experiment a preꢀinjection of 1a (0.3 mg/kg, ip), a selective GABAB agonist4, significantly
reduced (~35%) the uptake of [18F]1b in the whole brain of CDꢀ1 mice. This study demonstrated that the
binding of [18F]1b is specific in the mouse brain and, at least, partially mediated by the GABAB receptors.
Noteworthy, that at a higher dose of blocker (1 mg/kg) the tracer uptake increased (not shown). This is a
common observation in the cases when the binding site is present in the periphery and injection of blocker
increases the blood input function of radiotracers. Expression of GABAB in various peripheral organs is
well documented.31
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The demonstrated specific brain uptake of [18F]1b in mice are below the conventional 50% threshold for a
good PET tracer. Nevertheless, previous in vitro GABAB autoradiography showed a substantial species
difference. There was greater GABAB density (Bmax) in the nonꢀhuman primates (50ꢀ437 fmol/mg
tissue32) and human brain (60–700 fmol/mg tissue33, 34) than that in rodents (52 fmol/g tissue27). These
numbers have to be used carefully because different research groups using different experimental
protocols obtained them. Still, the species difference is encouraging and the greater GABAB levels in the
primates may justify further PET studies with [18F]1b and development of new GABAB PET tracers with
improved GABAB binding affinity and BBB permeability.
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baseline
block 0.3 mg/kg
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*
Figure 5. Blockade of [18F]1b accumulation in the whole brain of CD1 mice by injection of the selective
GABAB agonist 1a (0.3 mg/kg, ip). Time point – 30 min post radiotracer injection. The data are the mean
% injected dose/g tissue ± SD (n=5). The data show that administration of 1a significantly reduces the
whole brain uptake (*P < 0.01, ANOVA).
Experimental
All reagents were used directly as obtained commercially unless otherwise noted. Reaction progress
was monitored by thinꢀlayer chromatography (TLC) using silica gel 60 F254 (0.040−0.063 mm) with
detection by UV. All moistureꢀsensitive reactions were performed under an argon atmosphere using ovenꢀ
dried glassware and anhydrous solvents. Column flash chromatography was carried out using BDH silica
gel 60Å (40−63 micron). Analytical TLC was performed on plastic sheets coated with silica gel 60 F254
(0.25 mm thickness, E. Merck, Darmstadt, Germany). H and 13CꢀNMR spectra were recorded with a
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Brukerꢀ500 NMR spectrometer at nominal resonance frequencies of 500 MHz in CDCl3, CD3OD or
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DMSOꢀd6 (referenced to internal Me4Si at
δ 0 ppm). The chemical shifts (δ) were expressed in parts per
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million (ppm). Highꢀresolution mass spectra were recorded utilizing electrospray ionization (ESI) at the
University of Notre Dame Mass Spectrometry facility. All compounds that were tested in the biological
assays were analysed by combustion analysis (CHN) to confirm a purity of >95%. A dose calibrator
(Capintec 15R) was used for all radioactivity measurements. Radiolabeling was performed with a
modified GE MicroLab radiochemistry module. HPLC purification and analysis of radiolabeled
compounds were performed with Agilent 1260 Infinity System with UV detector and a Bioscan Flowꢀ
Count interface with a NaI radioactivity detector. The experimental animal protocols were approved by
the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.
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Chemistry
(R)-4-(4-chlorophenyl)pyrrolidin-2-one (5): To Rꢀbaclofen (Rꢀ1) (1 g, 4.68 mmol) was added 10 mL of
acetic acid. The reaction mixture was refluxed overnight. The mixture was cooled to room temperature,
the acetic acid was removed in vacuo and the resultant mixture treated with water (100 mL). An offꢀwhite
product precipitated and was filtered, washed with hexane and dried in vacuo to provide the (R)ꢀ4ꢀ(4ꢀ
1
chlorophenyl)pyrrolidinꢀ2ꢀone as a white solid (0.9 g, 99.3% yield). H NMR (500 MHz, CD3OD)
δ
7.33–7.22 (m, 4H), 3.79–3.64 (m, 2H), 3.39–3.32 (m, 1H), 2.73–2.66 (dd, J = 16.8, 8.8 Hz, 1H), 2.47–
2.40 (dd, J = 16.8, 8.8 Hz, 1H).
(R)-4-(4-chloro-3-nitrophenyl)pyrrolidin-2-one (6): (R)ꢀ4ꢀ(4ꢀchlorophenyl)pyrrolidinꢀ2ꢀone 5 (0.9 g,
4.6 mmol) was treated with sulfuric acid (5 mL). This solution was cooled in ice. Guanidine nitrate (0.56
g, 4.6 mmol) was added in portions. The reaction mixture was warmed to room temperature and stirred
for 1 h. The mixture was then poured into ice water and extracted with ethyl acetate. The organic layer
was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue
was purified by silica gel column chromatography (CH2Cl2: MeOH = 9:1) to give (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ
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nitrophenyl)pyrrolidinꢀ2ꢀone as a yellow solid (1.05 g, 95.4% yield). 1H NMR (500 MHz, CDCl3)
δ 7.77
5
6
(s, 1H), 7.55 (d, J = 10.0 Hz, 1H), 7.44 (d, J = 10.0 Hz, 1H), 6.13 (s, 1H), 3.86 (t, J = 10.0 Hz, 1H), 3.80–
7
8
3.76 (m, 1H), 3.44–3.41 (m, 1H), 2.84–2.79 (m, 1H), 2.49–2.44 (m, 1H).
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(R)-4-(3-amino-4-chlorophenyl)pyrrolidin-2-one (7): To
a
mixture of (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ
nitrophenyl)pyrrolidinꢀ2ꢀone 6 (1.0 g, 4.15 mmol), and NH4Cl (2.21 g, 41.5 mmol) in THF/MeOH/H2O
(10:5:3) (20 mL), was added Zn dust (2.71 g, 41.5 mmol) at 90 ˚C, then the mixture was refluxed for 1 h.
After completion of the reaction, the reaction mixture was filtered through Celite and partitioned between
EtOAc and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (CH2Cl2:
MeOH = 9:1) to give (R)ꢀ4ꢀ(3ꢀaminoꢀ4ꢀchlorophenyl)pyrrolidinꢀ2ꢀone as a pale yellow solid (0.85 g,
1
97.1% yield). H NMR (500 MHz, CD3OD)
δ 7.12–7.11 (d, J = 8.4 Hz, 1H), 6.74–6.73 (d, J = 2.0 Hz,
1H), 6.55–6.52 (m, 1H), 3.74–3.70 (m, 1H), 3.62–3.54 (m, 1H), 3.34–3.29 (m, 1H), 2.69–2.63 (dd, J =
16.4, 8.8 Hz,1H), 2.43–2.37 (dd, J = 16.4, 8.4 Hz, 1H).
(R)-4-(4-chloro-3-iodophenyl)pyrrolidin-2-one (8): To (R)ꢀ4ꢀ(3ꢀaminoꢀ4ꢀchlorophenyl)pyrrolidinꢀ2ꢀ
one 7 (0.8 g, 3.80 mmol) was added a mixture of 12N HCl (20 mL) and cooled to 0 °C. Sodium nitrite
(0.29 g, 4.17 mmol) in 10 mL water was added dropwise. The mixture was stirred for 30 minutes.
Potassium iodide (5.35 g, 32.3 mmol) in 50 mL of water was added dropwise at 0 °C. The mixture was
stirred for 1h at 0 °C and then 45 minutes at room temperature. The reaction mixture was then partitioned
between EtOAc and washed with 10% sodium thiosulfate, followed by water and brine. The organic layer
was separated, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue
was purified by silica gel column chromatography (CH2Cl2: MeOH = 9.5:0.5) to give (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ
1
iodophenyl)pyrrolidinꢀ2ꢀone as a pale yellow solid (1.0 g, 82.0% yield). H NMR (500 MHz, CDCl3)
δ
7.74 (s, 1H), 7.41 (d, J = 10.0 Hz, 1H), 7.19 (d, J = 10.0 Hz, 1H), 6.88 (s, 1H), 3.79 (t, J = 10.0 Hz, 1H),
3.64–3.61 (m, 1H), 3.40–3.36 (m, 1H), 2.77–2.71 (m, 1H), 2.46–2.41 (m, 1H).
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Tert-butyl (R)-4-(4-chloro-3-iodophenyl)-2-oxopyrrolidine-1-carboxylate (9): To the compound (R)ꢀ
4ꢀ(4ꢀchloroꢀ3ꢀiodophenyl)pyrrolidinꢀ2ꢀone 8 (1.0 g, 3.11 mmol) in CH2Cl2 (10 mL) was added DMAP
(0.037 g, 0.031 mmol), triethylamine (0.37 g, 3.73 mmol) and diꢀtertꢀbutyl dicarbonate (0.81 g, 3.73
mmol) at 0 °C, and then the mixture was stirred at room temperature for 12 h. After completion of the
reaction it was diluted with CH2Cl2 and washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. Flash chromatography of the residue over silica gel (hexane:EtOAc = 9.5:0.5)
afforded tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀiodophenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate as a pale yellow solid
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(1.21 g, 92.3% yield). 1H NMR (500 MHz, CDCl3)
δ 7.73 (s, 1H), 7.43 (d, J = 10.0 Hz, 1H), 7.17 (d, J =
10.0 Hz, 1H), 4.16–4.12 (m, 1H), 3.66–3.63 (m, 1H), 3.49–3.46 (m, 1H), 2.92–2.87 (m, 1H), 2.68–2.62
(m, 1H), 1.54 (s, 9H).
Tert-butyl (R)-4-(4-chloro-3-hydroxyphenyl)-2-oxopyrrolidine-1-carboxylate (10): To a solution of
tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀiodophenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 9 (1.2 g, 2.84 mmol) and
Bis(pinacolato)diboron (2.16 g, 8.53 mmol) in DMF (12.0 mL) was added Pd(dppf)Cl2.DCM (0.23 g,
0.028 mmol) and potassium acetate (1.11 g, 11.38 mmol). The reaction mixture was stirred at 90 °C for
12 h. After it cooled down to room temperature, to this solution, 35% hydrogen peroxide (12 mL) was
added dropwise and stirred overnight at room temperature. The mixture was diluted with saturated
ammonium chloride aqueous solution and extracted with dichloromethane (3 times). The combined
organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was
purified by column chromatography (Hexane/EtOAc = 3:7) to afford tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ
hydroxyphenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate as white solid (0.15 g, 17.0% yield). 1H NMR (500 MHz,
CDCl3)
δ 7.30 (d, J = 10.0 Hz, 1H), 6.91 (s, 1H), 6.75 (d, J = 10.0 Hz, 1H), 4.16–4.12 (m, 1H), 3.67–
3.64 (m, 1H), 3.50–3.46 (m, 1H), 2.91–2.86 (m, 1H), 2.69–2.64 (m, 1H), 1.54 (s, 9H).
(R)-4-amino-3-(4-chloro-3-(pyridin-4-ylmethoxy)phenyl)butanoic acid (1a): To a solution of tertꢀ
butyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀhydroxyphenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 10 (0.1 g, 0.32 mmol) in ethanol
(2 mL) was added K2CO3 (0.09 g, 0.64 mmol) followed by 4ꢀ(bromomethyl)pyridine hydrobromide
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(0.121 g, 0.48 mmol). The reaction mixture was heated overnight at 90 °C. The mixture was cooled to
room temperature and the solvent removed. The crude residue was treated with 6N HCl aqueous solution
(4 mL) and heated for 3 h at 90 °C. After that reaction mixture was cooled and adjusted to pH to 7 by
using aqueous NaHCO3 solution. The precipitate was filtered, washed with cold water and dried to give
(R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ(pyridinꢀ4ꢀylmethoxy)phenyl)butanoic acid as a pale brown solid (0.07 g,
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68.6% yield). 1H NMR (500 MHz, CD3OD)
δ 8.91 (d, J = 10.0 Hz, 2H), 8.26 (d, J = 10.0 Hz, 2H), 7.45
(d, J = 10.0 Hz, 1H), 7.18 (s, 1H), 7.03 (d, J = 10.0 Hz, 1H), 5.60 (s, 2H), 3.83–3.75 (m, 2H), 3.44–3.41
(m, 1H), 2.77–2.72 (m, 1H), 2.52–2.47 (m, 1H). 13C NMR (125 MHz, MeOD)
δ
178.3, 158.6, 153.2,
143.4, 141.8, 130.3, 124.2, 121.2, 120.9, 112.6, 68.0, 49.2, 39.9, 37.7. HRMS (ESI+) m/z calcd
[C16H18ClN2O3] [(M + H)]+ 321.1006, found 321.0987.
(R)-3-Acetyl-4-phenylmethyl-2-oxazolidinone (12): To a 250 mL round bottom flask equipped with a
stir bar, 10 g (56.43 mmol) of (R)ꢀ4ꢀ phenylmethylꢀ2ꢀoxazolidinone 11 was added, along with 100 mL of
THF under N2. The flask was then cooled to ꢀ78 °C, and 37.0 mL (59.30 mmol) of nꢀBuLi (1.6 M in
hexanes) was added. After 20 minutes 4.1 mL (57.50 mmol) of acetyl chloride was added dropwise. After
30 minutes the reaction mixture was allowed to warm to room temperature and stirred for 3h. After that
the reaction mixture was quenched with 100 mL of satd. aq. NH4Cl. The majority of the THF was
removed in vacuo. Methylene chloride was added and this solution was washed with 10% aq. NaOH. The
aqueous layer was extracted again with CH2Cl2 (2 x 30 mL). The combined organic layers were washed
with brine, dried with sodium sulfate, filtered, and concentrated in vacuo to yield (R)ꢀ3ꢀAcetylꢀ4ꢀ
1
phenylmethylꢀ2ꢀoxazolidinone as a white solid (11.0 g, 88.9% yield). H NMR (500 MHz, CDCl3) δ
7.35–7.33 (m, 2H), 7.29 (d, J = 10.0 Hz, 1H), 7.21 (d, J = 10.0 Hz, 2H), 4.69–4.66 (m, 1H), 4.19 (t, J =
10.0, 2H), 3.33 (d, J = 10.0, 1H), 2.78 (d, J = 10.0, 1H), 2.56 (s, 3H). 13C NMR (125 MHz, CDCl3)
δ
170.3, 153.7, 135.3, 129.4, 129.0, 127.4, 66.1, 55.0, 37.8, 23.8.
3-(Benzyloxy)-4-chlorobenzaldehyde (14): To a stirred solution of 4ꢀchloroꢀ3ꢀhydroxybenzaldehyde 13
(5.0 g, 31.93 mmol) in DMF (40.0 mL) was added benzyl bromide (6.55 g, 38.3 mmol) and K2CO3 (22.05
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g, 159.68 mmol). Then, the mixture was stirred at room temperature for 8h and then partitioned between
EtOAc and brine. The organic layer was separated, washed with brine, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (hexane:EtOAc = 1:9) to give 3ꢀ(benzyloxy)ꢀ4ꢀchlorobenzaldehyde as a white solid (7.4
g, 93.9% yield). 1H NMR (500 MHz, CDCl3) δ 9.92 (s, 1H), 7.56 (d, J = 5.0 Hz, 2H), 7.50–7.47 (m, 3H),
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7.42–7.39 (m, 3H), 7.35 (d, J = 10.0 Hz, 1H), 5.22 (s, 2H). 13C NMR (125 MHz, CDCl3)
δ 190.9, 154.9,
136.0, 135.8, 130.9, 130.3, 128.7, 128.3, 127.2, 124.4, 111.9, 70.9.
(E)-2-(benzyloxy)-1-chloro-4-(2-nitrovinyl)benzene (15): 3ꢀ(benzyloxy)ꢀ4ꢀchlorobenzaldehyde 14 (7.0
g, 28.37 mmol) was dissolved in acetic acid (8.5 mL), which was followed by the addition of
nitromethane (35.0 mL) and ammonium acetate (5.46 g, 70.94 mmol). The resulting solution was heated
to 105 °C for 90 minutes after which it was cooled and the solvent was evaporated under reduced
pressure. The residue was triturated with water (100 mL), resulting in a yellow precipitate, which was
filtered to obtain (E)ꢀ2ꢀ(benzyloxy)ꢀ1ꢀchloroꢀ4ꢀ(2ꢀnitrovinyl)benzene as a yellow solid (7.1 g, 86.0%
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yield). H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 15.0 Hz, 1H), 7.52–7.46 (m, 4H), 7.44–7.41 (m, 2H),
7.36 (d, J = 10.0 Hz, 1H), 7.11 (d, J = 10.0 Hz, 1H), 7.08 (d, J = 10.0 Hz, 1H), 5.21 (s, 2H). 13C NMR
(125 MHz, CDCl3)
70.9.
δ 154.8, 138.1, 137.4, 131.3, 128.8, 128.7, 128.4, 127.3, 127.2, 127.1, 126.6, 113.7,
(R)-4-benzyl-3-((R)-3-(3-(benzyloxy)-4-chlorophenyl)-4-nitrobutanoyl)oxazolidin-2-one (16):
A
solution of (R)ꢀ3ꢀacetylꢀ4ꢀbenzyloxazolidinꢀ2ꢀone (5.39 g, 24.06 mmol) in THF (70 mL) was cooled
o
under a nitrogen atmosphere to ꢀ78 C and a solution of NaHMDS (1M in THF, 26.95 mL, 25.77 mmol)
was added dropwise and stirred for 1h. A solution of nitroalkene 15 (7.0 g, 24.16 mmol) in THF (35 mL)
was added dropwise to the reaction solution and the mixture was stirred for a further hour. After
quenching with a saturated solution of ammonium chloride (100 mL), the mixture was extracted with
ethyl acetate (3 × 75 mL). The combined organic layers were washed with brine (100 mL), dried
(Na2SO4) and evaporated under reduced pressure. The resulting residue was purified by silica gel column
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chromatography (hexane:EtOAc = 1:9) to give (R)ꢀ4ꢀbenzylꢀ3ꢀ((R)ꢀ3ꢀ(3ꢀ(benzyloxy)ꢀ4ꢀchlorophenyl)ꢀ4ꢀ
nitrobutanoyl)oxazolidinꢀ2ꢀone as a pale yellow solid (6.5 g, 52.8% yield). 1H NMR (500 MHz, CDCl3) δ
7.47 (d, J = 5.0 Hz, 2H), 7.40 (t, J = 5.0 Hz, 2H), 7.38–7.32 (m, 4H), 7.29 (d, J = 10.0 Hz, 1H), 7.17 (d, J
= 10.0 Hz, 2H), 6.90 (s, 1H), 6.83 (d, J = 10.0 Hz, 1H), 5.16 (s, 2H), 4.70–4.66 (m, 1H), 4.61–4.55 (m,
2H), 4.15 (t, J = 5.0 Hz, 2H), 4.13–4.10 (m, 1H), 3.53–3.48 (m, 1H), 3.29–3.22 (m, 2H), 2.76–2.72 (m,
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1H). 13C NMR (125 MHz, CDCl3)
δ 169.8, 153.4, 138.4, 136.2, 134.9, 130.8, 129.4, 129.0, 18.6, 128.1,
127.3, 123.1, 120.5, 113.7, 79.3, 71.0, 66.5, 55.1, 39.3, 38.6, 37.8.
(R)-4-(3-(benzyloxy)-4-chlorophenyl)pyrrolidin-2-one (17): The oxazolidinone 16 (6 g, 11.78 mmol)
was dissolved in ethanol (110 mL), cooled to 0 °C and nickel chloride hexahydrate (2.80 g, 11.78 mmol)
was added, followed by the portionwise addition sodium borohydride (4.90 g, 129.67 mmol). The
resulting black suspension was stirred for two hours at 0°C and then allowed to warm to room
temperature over a further two hour period. Diluted hydrochloric acid solution (2 M, 30 mL) was added
slowly to the mixture and the excess solvent was evaporated under reduced pressure. The black residue
was dissolved with ethyl acetate (50 mL) and stirred with diluted hydrochloric acid solution (2 M, 30 mL)
until a colorless solution was obtained. The aqueous phase was extracted with ethyl acetate (2 × 50 mL),
and the combined organic layers were dried (Na2SO4) and evaporated under reduced pressure. The residue
was purified by flash column chromatography (SiO2, ethyl acetate/hexanes, 8/2 then ethyl
acetate/methanol, 9/1) to obtain (R)ꢀ4ꢀ(3ꢀ(benzyloxy)ꢀ4ꢀchlorophenyl)pyrrolidinꢀ2ꢀone as a colorless
1
solid (1.85 g, 52.1% yield over two steps). H NMR (500 MHz, CDCl3) δ 7.47 (d, J = 5.0 Hz, 2H), 7.40
(t, J = 5.0 Hz, 2H), 7.34 (d, J = 10.0 Hz, 2H), 6.83 (s, 1H), 6.80 (d, J = 10.0 Hz, 1H), 5.76 (s, 1H), 5.16 (s,
2H), 3.75 (t, J = 5.0 Hz, 2H), 3.65–3.62 (m, 1H), 3.34–3.31 (m, 1H), 2.74–2.68 (m, 1H), 2.44–2.39 (m,
1H). 13C NMR (125 MHz, CDCl3)
112.8, 70.9, 49.4, 40.0, 37.9.
δ 177.6, 154.3, 142.2, 136.3, 130.5, 128.6, 128.1, 127.2, 122.0, 119.9,
Tert-butyl (R)-4-(3-(benzyloxy)-4-chlorophenyl)-2-oxopyrrolidine-1-carboxylate (18): To the
compound (R)ꢀ4ꢀ(3ꢀ(benzyloxy)ꢀ4ꢀchlorophenyl)pyrrolidinꢀ2ꢀone 17 (1.5 g, 4.97 mmol) in CH2Cl2 (10
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mL) was added DMAP (0.60 g, 0.497 mmol), triethylamine (0.603 g, 5.96 mmol) and diꢀtertꢀbutyl
dicarbonate (1.30 g, 5.96 mmol) at 0 °C, and then, the mixture was stirred at room temperature for 8h.
After completion of the reaction diluted with CH2Cl2 and washed with brine, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Flash chromatography of the residue over silica gel
(hexane:EtOAc = 8:2) afforded tertꢀbutyl (R)ꢀ4ꢀ(3ꢀ(benzyloxy)ꢀ4ꢀchlorophenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀ
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carboxylate as a colorless solid (1.8 g, 90.4% yield). H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 5.0 Hz,
2H), 7.39 (t, J = 5.0 Hz, 2H), 7.36–7.31 (m, 2H), 6.81 (s, 1H), 6.78 (d, J = 10.0 Hz, 1H), 5.16 (s, 2H),
4.14–4.10 (m, 1H), 3.63–3.59 (m, 1H), 3.48–3.44 (m, 1H), 2.88–2.83 (m, 1H), 2.65–2.59 (m, 1H), 1.54
(s, 9H). 13C NMR (125 MHz, CDCl3)
δ 172.6, 154.4, 149.8, 140.5, 136.2, 130.7, 128.7, 128.2, 127.2,
122.5, 119.8, 112.8, 83.3, 71.0, 52.9, 40.2, 36.2, 28.0.
Tert-butyl (R)-4-(4-chloro-3-hydroxyphenyl)-2-oxopyrrolidine-1-carboxylate (10): The compound 18
(1.60 g, 3.98 mmol) was dissolved in methanol (16 mL). Palladium hydroxide (0.32 g) on activated
charcoal (20 wt%) was added to the suspension, flushed and pressurized with hydrogen (50 psi) and
stirred for 30 minutes at room temperature. The reaction mixture was filtered through a Celite pad, and
the filtrate was concentrated under reduced pressure. The residue was purified by flash column
chromatography (ethyl acetate/hexanes) to obtain tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀhydroxyphenyl)ꢀ2ꢀ
oxopyrrolidineꢀ1ꢀcarboxylate as a colorless solid (1.0 g, 80.0% yield). 1H NMR (500 MHz, CDCl3)
δ 7.30
(d, J = 10.0 Hz, 1H), 6.91 (s, 1H), 6.75 (d, J = 10.0 Hz, 1H), 4.16–4.12 (m, 1H), 3.67–3.64 (m, 1H), 3.50–
13
3.46 (m, 1H), 2.91–2.86 (m, 1H), 2.69–2.64 (m, 1H), 1.54 (s, 9H). C NMR (125 MHz, CDCl3)
151.7, 149.8, 141.4, 129.5, 119.6, 118.9, 114.7, 83.3, 52.8, 40.1, 35.9, 28.0.
δ 172.7,
Tert-butyl (R)-4-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)-2-oxopyrrolidine-1-carboxylate
(19a): To a mixture of tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀhydroxyphenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 10
(0.2 g, 0.64 mmol) and PPh3 (0.25 g, 0.96 mmol) in THF (10 mL) at 0 °C was added DIAD (0.196 g, 0.96
mmol). After stirring for 1 h, (2ꢀfluoropyridinꢀ4ꢀyl)methanol (0.085 g, 0.67 mmol) was added to the
reaction mixture 0 °C. The reaction mixture was stirred for 2 h at 0 °C and overnight at room temperature.
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The resulting mixture was partitioned between ethyl acetate and water. The aqueous layer was extracted
with ethyl acetate and the combined organic layers were dried (Na2SO4) and evaporated under reduced
pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes) to obtain tertꢀ
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butyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀfluoropyridinꢀ4ꢀyl)methoxy)phenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate as
a
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colorless solid (0.2 g, 76.9% yield). H NMR (500 MHz, CDCl3)
δ 8.28 (d, J = 5.0 Hz, 1H), 7.43 (d, J =
10.0 Hz, 1H), 7.32 (d, J = 5.0 Hz, 1H), 7.13 (s, 1H), 6.91 (s, 1H), 6.88 (d, J = 5.0 Hz, 1H), 6.80 (s, 1H),
5.20 (s, 2H), 4.19–4.15 (m, 1H), 3.69–3.65 (m, 1H), 3.53–3.50 (m, 1H), 2.95–2.90 (m, 1H), 2.71–2.65
(m, 1H), 1.56 (s, 9H). 13C NMR (125 MHz, CDCl3)
δ 172.3, 153.7, 149.8, 148.1, 148.0, 141.0, 131.1,
122.6, 120.7, 118.8, 112.4, 107.3, 106.9, 83.4, 68.6, 52.9, 40.2, 36.1, 28.0.
Tert-butyl (R)-4-(4-chloro-3-((3-fluoropyridin-4-yl)methoxy)phenyl)-2-oxopyrrolidine-1-carboxylate
(19b): To a mixture of tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀhydroxyphenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 10
(0.2 g, 0.64 mmol) and PPh3 (0.25 g, 0.96 mmol) in THF (10 mL) at 0 °C was added DIAD (0.196 g, 0.96
mmol). After stirring for 1 h, (3ꢀfluoropyridinꢀ4ꢀyl)methanol (0.085 g, 0.67 mmol) was added to the
reaction mixture 0 °C. The reaction mixture was stirred for 2 h at 0 °C and overnight at room temperature.
The resulting mixture was partitioned between ethyl acetate and water. The aqueous layer was extracted
with ethyl acetate and the combined organic layers were dried (Na2SO4) and evaporated under reduced
pressure. The residue was purified by flash column chromatography (ethyl acetate/hexanes) to obtain tertꢀ
butyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ((3ꢀfluoropyridinꢀ4ꢀyl)methoxy)phenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate as
colorless solid (0.2 g, 76.9% yield). 1H NMR (500 MHz, CDCl3)
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δ
8.55 (s, 2H), 7.78 (s, 1H), 7.43 (d, J =
5.0 Hz, 1H), 6.91 (s, 1H), 6.88 (d, J = 5.0 Hz, 1H), 5.29 (s, 2H), 4.20–4.16 (m, 1H), 3.70–3.66 (m, 1H),
3.55–3.52 (m, 1H), 2.96–2.91 (m, 1H), 2.72–2.68 (m, 1H), 1.56 (s, 9H). 13C NMR (125 MHz, CDCl3)
δ
172.3, 157.6, 153.6, 149.8, 144.3, 141.1, 131.1, 131.0, 123.4, 122.6, 120.9, 119.7, 112.6, 83.4, 63.4, 52.8,
40.3, 36.2, 28.1.
Tert-butyl
(R)-4-(3-((2-bromopyridin-4-yl)methoxy)-4-chlorophenyl)-2-oxopyrrolidine-1-
carboxylate (19c): To a mixture of tertꢀbutyl (R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀhydroxyphenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀ
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carboxylate 10 (0.2 g, 0.64 mmol) and PPh3 (0.25 g, 0.96 mmol) in THF (10 mL) at 0 °C was added
DIAD (0.196 g, 0.96 mmol). After stirring for 1 h, (2ꢀbromopyridinꢀ4ꢀyl)methanol (0.126 g, 0.67 mmol)
was added to the reaction mixture 0 °C. The reaction mixture was stirred for 2 h at 0 °C and overnight at
room temperature. The resulting mixture was partitioned between ethyl acetate and water. The aqueous
layer was extracted with ethyl acetate and the combined organic layers were dried (Na2SO4) and
evaporated under reduced pressure. The residue was purified by flash column chromatography (ethyl
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acetate/hexanes) to obtain tertꢀbutyl (R)ꢀ4ꢀ(3ꢀ((2ꢀbromopyridinꢀ4ꢀyl)methoxy)ꢀ4ꢀchlorophenyl)ꢀ2ꢀ
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oxopyrrolidineꢀ1ꢀcarboxylate as a colorless solid (0.26 g, 84.1% yield). H NMR (500 MHz, CDCl3)
δ
8.43 (d, J = 5.0 Hz, 1H), 7.66 (s, 1H), 7.44–7.40 (m, 2H), 6.90 (d, J = 5.0 Hz, 1H), 6.79 (s, 1H), 5.15 (s,
2H), 4.19–4.15 (m, 1H), 3.69–3.65 (m, 1H), 3.53–3.50 (m, 1H), 2.95–2.90 (m, 1H), 2.71–2.65 (m, 1H),
1.56 (s, 9H). 13C NMR (125 MHz, CDCl3)
δ 172.4, 153.7, 150.4, 149.7, 148.4, 142.7, 141.0, 131.0, 125.5,
122.5, 120.7, 120.3, 112.6, 83.3, 68.4, 52.9, 40.2, 36.2, 28.0.
(R)-4-amino-3-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1b): To tertꢀbutyl
(R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀfluoropyridinꢀ4ꢀyl)methoxy)phenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 19a (0.1 g,
2.37 mmol) was added 3 mL of HCl in 1,4ꢀdioxane. The reaction mixture was refluxed overnight. The
mixture was cooled to room temperature and adjusted to pH 7 by using aqueous NaHCO3 solution. The
precipitate was filtered, washed with cold water and dried to give (R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀ
1
fluoropyridinꢀ4ꢀyl)methoxy)phenyl)butanoic acid as a white solid (0.03 g, 37.5% yield). H NMR (500
MHz, CDCl3)
δ 8.26 (d, J = 10.0 Hz, 1H), 7.81(s, 1H), 7.59–7.56 (m, 1H), 7.46–7.40 (m, 2H), 7.30 (d, J
= 10.0 Hz, 1H), 7.22 (d, J = 10.0 Hz, 1H), 7.11–7.06 (m, 1H), 4.68 (s, 1H), 3.64 (d, J = 10.0 Hz, 1H),
1.43–1.40 (m, 1H), 3.28 (d, J = 5.0 Hz, 1H), 2.32–2.29 (m, 1H), 1.84–1.82 (m, 1H), 1.76–1.73 (m, 2H),
1.72 (s, 9H), 1.60 (s, 1H), 1.54–1.47 (m, 1H), 1.43–1.37 (m, 1H), 1.28 (s, 3H), 1.15 (s, 3H), 1.09 (d, J =
10.0 Hz, 3H). 13C NMR (125 MHz, MeOD)
δ 178.4, 165.0, 153.6, 147.3, 147.2, 143.1, 130.2, 121.2,
120.3, 119.3, 112.6, 106.9, 68.0, 49.2, 39.9, 37.7. HRMS (ESI+) m/z calcd [C16H17ClFN2O3] [(M + H)]+
339.0912, found 339.0906.
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(R)-4-amino-3-(4-chloro-3-((3-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1c): To tertꢀbutyl
(R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ((3ꢀfluoropyridinꢀ4ꢀyl)methoxy)phenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 19b (0.1 g,
2.37 mmol) was added 3 mL of 6N HCl solution. The reaction mixture was refluxed overnight. The
mixture was cooled to room temperature and adjusts the pH to 7 by using aqueous NaHCO3 solution. The
precipitate was filtered, washed with cold water and dried to give (R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ((3ꢀ
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fluoropyridinꢀ4ꢀyl)methoxy)phenyl)butanoic acid as a white solid (0.03 g, 37.5% yield). H NMR (500
MHz, CDCl3)
δ 8.26 (d, J = 10.0 Hz, 1H), 7.81(s, 1H), 7.59–7.56 (m, 1H), 7.46–7.40 (m, 2H), 7.30 (d, J
= 10.0 Hz, 1H), 7.22 (d, J = 10.0 Hz, 1H), 7.11–7.06 (m, 1H), 4.68 (s, 1H), 3.64 (d, J = 10.0 Hz, 1H),
1.43–1.40 (m, 1H), 3.28 (d, J = 5.0 Hz, 1H), 2.32–2.29 (m, 1H), 1.84–1.82 (m, 1H), 1.76–1.73 (m, 2H),
1.72 (s, 9H), 1.60 (s, 1H), 1.54–1.47 (m, 1H), 1.43–1.37 (m, 1H), 1.28 (s, 3H), 1.15 (s, 3H), 1.09 (d, J =
10.0 Hz, 3H). 13C NMR (125 MHz, MeOD)
δ 178.4, 153.6, 145.4, 143.2, 137.0, 136.8, 134.0, 130.2,
123.3, 121.3, 120.4, 112.7, 63.1, 49.2, 39.9, 37.7. HRMS (ESI+) m/z calcd [C16H17ClFN2O3] [(M + H)]+
339.0912, found 339.0906.
(R)-4-amino-3-(4-chloro-3-((2-bromopyridin-4-yl)methoxy)phenyl)butanoic acid (1d): To tertꢀbutyl
(R)ꢀ4ꢀ(4ꢀchloroꢀ3ꢀ((2ꢀbromopyridinꢀ4ꢀyl)methoxy)phenyl)ꢀ2ꢀoxopyrrolidineꢀ1ꢀcarboxylate 19c (0.2 g,
0.415 mmol) was added 4 mL of 55% HBr solution. The reaction mixture was refluxed overnight. The
mixture was cooled to room temperature and adjusted to pH 7 by using aqueous NaHCO3 solution. The
precipitate was filtered, washed with cold water and dried to give (R)ꢀ4ꢀaminoꢀ3ꢀ(4ꢀchloroꢀ3ꢀ((3ꢀ
1
bromopyridinꢀ4ꢀyl)methoxy)phenyl)butanoic acid as a white solid (0.12 g, 72.7% yield). H NMR (500
MHz, CDCl3)
δ 8.26 (d, J = 10.0 Hz, 1H), 7.81(s, 1H), 7.59–7.56 (m, 1H), 7.46–7.40 (m, 2H), 7.30 (d, J
= 10.0 Hz, 1H), 7.22 (d, J = 10.0 Hz, 1H), 7.11–7.06 (m, 1H), 4.68 (s, 1H), 3.64 (d, J = 10.0 Hz, 1H),
1.43–1.40 (m, 1H), 3.28 (d, J = 5.0 Hz, 1H), 2.32–2.29 (m, 1H), 1.84–1.82 (m, 1H), 1.76–1.73 (m, 2H),
1.72 (s, 9H), 1.60 (s, 1H), 1.54–1.47 (m, 1H), 1.43–1.37 (m, 1H), 1.28 (s, 3H), 1.15 (s, 3H), 1.09 (d, J =
10.0 Hz, 3H). 13C NMR (125 MHz, MeOD)
δ 178.4, 153.6, 150.3, 149.8, 143.1, 141.6, 130.2, 125.6,
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121.2, 120.8, 120.4, 112.6, 67.7, 49.2, 39.9, 37.7. HRMS (ESI+) m/z calcd [C16H17BrClN2O3] [(M + H)]+
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399.0111, found 399.0106.
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In vitro GABAB assay
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The in vitro assay of compounds 1a-1c and Rꢀbaclofen (Rꢀ1) was performed commercially by Eurofinsꢀ
CEREP.
GABAꢀB agonist assay. In brief, the human GABAB receptor transfected CHO cells were seeded onto a
96ꢀwell plate at a density of 8.104 cells/well in HBSS buffer + 20 mM HEPES and 0.1% BSA and were
allowed to equilibrate for 60 min at 28°C before the start of the experiment. The assay was done in
duplicate in two independent experiments. The system measured changes in electric impedance that occur
in each well upon stimulation of cell surface receptors. Plates were placed onto the system and
measurements are made at a temperature of 28 °C. Solutions are added simultaneously to all 96 wells
using an integrated fluidics system: HBSS (basal control), reference agonist at 100 µM (stimulated
control), reference agonist (EC50 determination) or the test compounds. Impedance measurements are
monitored for 10 minutes after ligand addition. The standard reference agonist is 3ꢀAPMPA, which was
tested in each experiment at several concentrations to generate a concentrationꢀresponse curve from
which its EC50 value is calculated.
GABAꢀB radioligand binding assay (Eurofins catalog number 228510). Source: Wistar Rat brain.
Radiotracer: [3H]CGPꢀ54626, concentration 0.60 nM, Kd = 2.3 nM, Bmax = 1.1 pmole/mg protein.
Vehicle: 1% aq. DMSO. Nonꢀspecific ligand: CGPꢀ54626, concentration 100 µM. Incubation conditions:
20 min at 25oC. Incubation buffer: 50 mM TrisꢀHCl, pH 7.4, 2.5 mM CaCl2. The assay was done in
duplicate in two independent experiments at the concentration of test compound of 100 nM. Mean %
inhibition was calculated.
Radiosynthesis of [18F]1b.
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A solution of the [18F]fluoride prepared with a PETTrace cyclotron (General Electric; GE), 10 mg of
Kryptofix 222®, and 1.6 mg of K2C2O4 in 1 mL of 50% aqueous acetonitrile was added to a reaction
vessel of a GE MicroLab module (Milwaukee, WI). The mixture was heated at 120ꢀ135 °C under a
stream of argon, while water was evaporated azeotropically after the additions of 2 mL of CH3CN. A
solution of the corresponding bromo precursor 1d (2 mg) in anhydrous DMSO (0.8 mL) was added to the
reaction vessel and heated at 180 °C for 10 min. The reaction mixture was cooled, diluted with 0.7 mL of
water and injected onto the reverseꢀphase semiꢀpreparative HPLC: column Luna C18 10x250 mm, 10
micron (Phenomenex); mobile phase 32% acetonitrile: 68% 0.1M aqueous ammonium formate, 10
ml/min. The radioactive peak with a retention time of 15.5 min was collected in 50 mL of water. The
aqueous solution was transferred through an activated Waters Cꢀ18 SepꢀPak Plus (Milford, MA) solidꢀ
phase extraction (SPE) cartridge. After washing the SPE with 10 mL saline, the product was eluted with a
1 mL of ethanol into a collection vial and 10 mL of 0.9% saline was added through the same SPE. The
final product [18F]1b was then analyzed by analytical HPLC using a UV detector at 254 nm to determine
the radiochemical purity and molar radioactivity at the time the synthesis ended. The total synthesis time
including QC was 65 min.
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Analytical HPLC conditions: XBridge, 10 micron, 4.6 x 250 mm; mobile phase: 40:60 (acetonitrile: 0.1M
aqueous ammonium formate); flow rate 3 mL/min; UV ꢀ 254 nm; retention time 3.1 min.
Biodistribution studies of [18F]1b in CD-1 mice
Baseline study: Male, CDꢀ1 mice weighing 25ꢀ30 g from Charles River Laboratories, (Wilmington,
MA) were used for biodistribution studies. The animals were sacrificed by cervical dislocation at various
times following injection of [18F]1b (100 µCi, molar radioactivity 7200 mCi/ꢁmol, in 0.2 mL saline) into
a lateral tail vein; six animals per time point. The brains were rapidly removed and dissected on ice. The
brain regions of interest were weighed and their radioactivity content was determined in an automated γꢀ
counter with a counting error below 3%. Aliquots of the injectate were prepared as standards and their
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radioactivity content was counted along with the tissue samples; the percent of injected dose per gram of
tissue (%ID/g tissue) was calculated. All experimental protocols were approved by the Animal Care and
Use Committee of the Johns Hopkins Medical Institutions.
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Blocking study: In vivo GABAB receptor blocking studies were carried out in CDꢀ1 male mice (5 per
group) by intraperitoneal (i.p.) administration of GABAB agonist 1a (0.3 mg/kg) followed by i.v. injection
of the radiotracer [18F]1b (100 µCi, molar radioactivity ~6400 mCi/ꢀmol, 0.1 mL, saline solution at pH =
5) 15 min thereafter. Control animals were injected with 0.1 mL of saline at pH = 5. Thirty min after the
administration of [18F]1b brain tissues were harvested, and their radioactivity content was determined.
Conclusion
In this study we synthesized GABAB agonists 1b-c for PET imaging of GABAB in animals. The most
potent agonist with greatest inhibition binding affinity 1b was radiolabeled with 18F with good
radiochemical yield, high radiochemical purity and high molar radioactivity. The mouse experiments with
the PET radiotracer [18F]1b demonstrated the feasibility of ex vivo quantification of GABAB receptors.
The future research on GABAB PET imaging should target radiotracers with improved specific binding
and greater bloodꢀbrain barrier permeability.
AUTHOR INFORMATION
Corresponding Author
*Phone: (1) 410ꢀ614ꢀ5130; eꢀmail: ahorti1@jhmi.edu.
Author Contributions
Ravi Naik has synthesized all unlabeled compounds.
Andrew G. Horti and Dean F. Wong designed the experiments.
Andrew G. Horti and Dean F. Wong analyzed the animal data.
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Andrew G. Horti and Ravi Naik were involved in writing the manuscript.
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Andrew G. Horti and Robert F. Dannals were involved in radiosynthesis of GABAB radiotracer.
Heather Valentine, Andrew G. Horti, Ravi Naik were involved in the mouse studies.
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Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This research was supported in part by SFARI (Simon Foundation Autism Research Initiative) grant
(Wong, Horti) and Division of Nuclear Medicine and Molecular Imaging. We thank Paige Finley for the
help with animal experiments and Julia Buchanan for editorial comments.
We thank Dr. Thomas Morley of Invicro (Boston, MA) for useful synthetic procedures related to the
synthesis of compound 17.
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