Synthesis and Preliminary Evaluation of [11C]GNE-1023 as a Potent PET Probe for Imaging Leucine-Rich Repeat Kinase 2 (LRRK2) in Parkinson's Disease

Article abstract of DOI:10.1002/cmdc.201900321

Leucine-rich repeat kinase 2 (LRRK2) is a large protein involved in the pathogenesis of Parkinson's disease (PD). It has been demonstrated that PD is mainly conferred by LRRK2 mutations that bring about increased kinase activity. As a consequence, selecti

Full text of DOI:10.1002/cmdc.201900321

Accepted Article
Title: Synthesis and preliminary evaluation of [11C]GNE-1023 as a
novel potent PET probe for imaging leucine-rich repeat kinase 2
(LRRK2) in Parkinson’s disease
Authors: Zhen Chen, Tuo Shao, Wei Gao, Hualong Fu, Thomas Lee
Collier, Jian Rong, Xiaoyun Deng, Qingzhen Yu, Xiaofei
Zhang, April T. Davenport, James B. Daunais, Hsiao-Ying
Wey, Yihan Shao, Lee Josephson, Wen-Wei Qiu, and Steven
Liang
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To be cited as: ChemMedChem 10.1002/cmdc.201900321
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900321
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10.1002/cmdc.201900321
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Synthesis and preliminary evaluation of [11C]GNE-1023 as a novel
potent PET probe for imaging leucine-rich repeat kinase 2 (LRRK2)
in Parkinson’s disease
Zhen Chen,[a]+ Tuo Shao,[a]+ Wei Gao[b] Hualong Fu,[a] Thomas Lee Collier,[a] Jian Rong,[a] Xiaoyun
Deng,[a] Qingzhen Yu,[a] Xiaofei Zhang,[a] April T. Davenport,[c] James B. Daunais,[c] Hsiao-Ying Wey,[d]
Yihan Shao,[e] Lee Josephson,[a] Wen-Wei Qiu*[b] and Steven Liang*[a]
[a]
[b]
Dr. Z. Chen, Dr. T. Shao, Dr. H. Fu, Dr T. L. Collier, Dr. J. Rong, Dr. X. Deng, Dr. Q. Yu, Dr. X. Zhang, Prof. Dr. L. Josephson, Prof. Dr. S. Liang
Department of Radiology, Division of Nuclear Medicine and Molecular Imaging
Massachusetts General Hospital and Harvard Medical School
55 Fruit Street, Boston, Massachusetts 02114, United States
E-mail: liang.steven@mgh.harvard.edu
W. Gao, Prof. Dr. W.-W. Qiu
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering
East China Normal University
500 Dongchuan Road, Shanghai, 200241, China
E-mail: wwqiu@chem.ecnu.edu.cn
[c]
[d]
[e]
A. T. Davenport, Prof. Dr. J. B. Daunais
Department of Physiology and Pharmacology
Wake Forest School of Medicine
Winston Salem, North Carolina 27157, United States
Prof. Dr. H.-Y. Wey
Athinoula A Martinos Center for Biomedical Imaging, Department of Radiology
Massachusetts General Hospital and Harvard Medical School
55 Fruit Street, Boston, Massachusetts 02114, United States
Prof. Dr. Y. Shao
Department of Chemistry and Biochemistry
University of Oklahoma
Norman, Oklahoma 73019, United States
+ These authors contribute equally to this work.
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: Leucin-rich repeat kinase 2 (LRRK2) is a large protein that
is involved in the pathogenesis of Parkinson’s disease (PD). It has
been demonstrated that LRRK2 mutations mainly confers PD, which
bring about increased kinase activity of LRRK2. As a consequence,
selective inhibition of LRRK2 may help to recover the normal functions
of LRRK2, and thereafter serve as an alternatively promising
therapeutic target for PD treatment. The mapping of LRRK2 with PET
studies allows a deep understanding of PD and other LRRK2-related
disorders, as well as helps to validate and translate novel LRRK2
inhibitors. However, to date no LRRK2 PET probes have been
reported in the primary literature. In this study, we present a facile
synthesis and preliminary evaluation of [11C]GNE-1023 as a novel
potent PET probe for LRRK2 imaging in PD. [11C]GNE-1023 was
synthesized in good radiochemical yield (10% non-decay corrected
RCY), excellent radiochemical purity (>99%) and high molar activity
(>37 GBq / μmol). Excellent in vitro binding specificity of [11C]GNE-
1023 towards LRRK2 was demonstrated in cross species studies,
including rat and nonhuman primate brain tissues by autoradiography
of 60.[1] Despite great efforts devoted to the investigation of PD
pathogenesis, currently the detailed etiology still remains unclear,
and no effective therapy has been approved to prevent, cure and
slow the PD progress. In the past decades, the leucine-rich kinase
2 (LRRK2) gene has been established as being involved in PD
pathogenesis. The LRRK2 gene is a large protein comprising a
central enzymatic core and
a diversity of protein-protein
interaction domains[2-4], and is widely distributed throughout the
CNS and other peripheral organs. While, in rodent brain, LRRK2
exhibits regional heterogeneity in expression levels, and the
highest levels are found in the striatum, cerebral cortex and
hippocampus,[5] in human brain, LRRK2 is mostly expressed in
the putamen and substantia nigra.[6] LRRK2 is also expressed in
the lung, kidney, liver and heart.[7] It has been demonstrated that
mutations of LRRK2 are associated with PD, which are linked to
~10% of familial cases and 2% of sporadic cases.[6, 8-10] Numerous
in vitro and in vivo studies indicated that multiple mutations of
LRRK2 can lead to increased kinase activity, and the most
common mutation is dominated by a G2019S variant.[11-16] As a
consequence, selective inhibition of LRRK2 may help to recover
the normal functions of LRRK2, and thereafter serve as a
promising therapeutic target for PD treatment.[17-18] In the last
decade, several LRRK2 inhibitors including GNE-1023, GNE-
7915, GNE-0877, GNE-9605, PF-06447475 and MLi-2 have been
reported (Figure 1).[19-23] Among these, GNE-1023 [3-methoxy-4-
((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-
experiments.
Subsequent whole-body biodistribution studies
indicated limited brain uptake and urinary and hepatobiliary
elimination of this radioligand. This study may pave the way for the
further development of a new generation of LRRK2 PET probes.
Parkinson’s disease (PD) is one of the most prevailing
neurodegenerative disorders in the central nervous system (CNS),
which affects approximately 10% of the population over the age
1
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yl)amino)phenyl)-(morpholino)meth-anone] was identified as a
potent, selective and brain-penetrant LRRK2 inhibitor (IC50 = 9
nM), which efficiently reduced the kinase activity of LRRK2 in
HEK293 cell assays as well as in the bacterial artificial
chromosome (BAC) transgenic mouse brain overexpressing
G2019S mutant LRRK2.[24] Particularly noteworthy is that this
compound and GNE-1023 entail a phenolic methyl group, which
would allow a facile labeling with carbon-11 for PET imaging
studies.[25]
proceeded under basic conditions to generate compound 5 in
moderate yield (40%). The desired LRRK2 inhibitor 6 was
obtained by SN2 displacement reaction of 5 with aniline 3 at 185
oC in a co-solvent of trifluoroacetic acid (TFA) and butanol (n-
BuOH). Ultimately, compound 6 was swiftly demethylated by
boron tribromide (BBr3) to deliver the corresponding phenolic
precursor 7 in 69% yield for subsequent radiosynthesis.
Scheme 1. Synthesis of LRRK2 inhibitor 6 (GNE-1023) and its demethylated
precursor 7. (i) SOCl2, DME, reflux,
6 h; (ii) morpholine, 3-methoxy-4-
nitrobenzoyl chloride, DIPEA, THF, 0 oC, 1h, 96% over two steps; (iii) Fe, AcOH,
r.t., 3 h, 90%; (iv) MeNH2, Et3N, MeOH, r.t., 12 h, 40%; (v) TFA, 2-butyl alcohol,
185 oC, 12 h, 31%; (vi) BBr3, DCM, r.t., 12 h, 69%. DME = 1,2-dimethoxyethane,
Figure 1. Structures of reported LRRK2 inhibitors.
As a non-invasive and highly-sensitive molecular imaging tool,
positron emission tomography (PET) has been widely used to
probe in vivo biological processes under normal or disease
conditions.[26-28] PET studies of LRRK2 would enable a deep
understanding of PD and other LRRK2-related disorders, as well
as help to validate and translate novel LRRK2 inhibitors. Despite
these benefits, to date no LRRK2-specific PET probe has been
reported in the primary literature. Therefore, an unmet clinical
need of LRRK2 imaging probes together with therapeutic
potential of LRRK2-related pharmacotherapy represents an
intensive motivation in the field. In 2013, several 11C- or 18F-
labeled LRRK2 inhibitors based on GNE-1023 and its close
analogs were disclosed in a patent of Genentech with limited
biological information.[29] To date, there is only one LRRK2 PET
ligand [11C]HG-10-102-01 developed by Zheng and co-workers in
2017 from the primary literature.[30] However, no biological data
was reported for this ligand. It is worth noting that, during the
review of this manuscript, Ding et al reported two 18F-labeled
LRRK2 inhibitors in a conference proceeding and biological
evaluation is underway.[31] Here we present an efficient synthesis
and preliminary in vitro and in vivo evaluation of [11C]GNE-1023
as a potential PET probe for LRRK2 imaging in PD.
DIPEA
=
N,N-diisopropylethylamine, THF
=
tetrahydrofuran, TFA
=
trifluoroacetic acid, DCM = dichloromethane.
Attributed to the existence of methoxy group in GNE-1023,
radiosynthesis of compound 6 can be conveniently performed
using [11C]CH3I from its corresponding phenolic precursor 7. All
processes from radiolabeling, purification and formulation of
[11C]6 were carried out with an automated synthetic module
(TRACERlab FX MeI, GE). The radiosynthesis of [11C]6 was
o
conducted by reacting 7 with [11C]CH3I at 80 C for 5 min in the
presence of NaOH in N,N-dimethylformide (DMF) (Scheme 2A).
It should be noted that the amount of precursor had a profound
effect on the RCY of this radiolabeling. While desired product was
generated with 1.0 mg precursor (Scheme 2A; entries 1 and 2),
increasing amount of precursor remarkably improved the reaction
yield (entries 3 and 4). In particular, a quantitative radiochemical
conversion with high isolated radiochemical yield (entry 4) was
achieved when 1.6 mg precursor was used. The reaction mixture
was purified with semi-preparative reverse high-performance
liquid chromatography (HPLC) to give [11C]6, which was
subsequently formulated in saline containing 10% of ethanol.
Ultimately, [11C]6 was obtained in an average of 10% non-decay
corrected radiochemical yield (RCY) relative to starting [11C]CO2
at the end of synthesis (EOS) with excellent radiochemical purity
(>99%) and high molar activity (>37 GBq/μmol). Average
synthetic time was 42 minutes. In addition, no radiolysis was
observed for [11C]6 up to 100 min after formulation (Scheme 2B).
We performed the synthesis of LRRK2 inhibitor 6 (GNE-1023)
and its corresponding demethylated precursor 7 using modified
procedures as depicted in Scheme 1. Briefly, commercially
available starting material 3-methoxy-4-nitrobenzoic acid 1 was
activated with SOCl2, and then coupled with morpholine to deliver
amide 2 in 96% yield over two steps. Iron-mediated reduction of
the nitro group in 2 occurred smoothly to yield aniline derivative 3
with high efficiency in the presence of acetic acid. In parellel,
amination reaction of commercially available starting material 2,4-
dichloro-5-(trifluoromethyl)pyrimidine 4 with methylamine readily
Lipophilicity is an emprical parameter to predict the ability of a
candidate to cross the blood-brain barrier (BBB), and favors a
range of 1.0-3.5.[32-34] The lipophilicity of [11C]6 (logD) was
determined to be 2.93 ± 0.02 through liquid-liquid partition
between PBS and n-octanol, which is also well-known as the
2
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COMMUNICATION
‘shake flask method’.[35] This result indicated that [11C]6
possesses a high possibility to penetrate the BBB.
Figure 2. In vitro autoradiography of [11C]6 in rat brain sections. (A) Brain
sections were treated with [11C]6; (B) Regional radioactivity distribution of [11C]6
in rat brain (baseline). The data were normalized to % of radioactivity vs that in
cerebellum (n = 3); (C) Brain sections were pre-treated with PF-06447475 (10
μM), a known LRRK2 inhibitor, followed by [11C]6; (D) Blocking studies. The
data were normalized to % of radioactivity vs control (n = 3). Hip = hippocampus;
Ctx = cerebral cortex; Str = striatum; Cb = cerebellum. Asterisks indicate
statistical significance. *p < 0.05, **p ≤0.01, and ***p ≤0.001.
Scheme 2. (A) Optimization of reaction parameters for radiosynthesis of [11C]6.
(i) [11C]MeI, DMF, 80 oC, 5min. a Non-decay corrected RCY relative to starting
[
11C]CO2. (B) Stability of radioligand [11C]6 in saline containing 10% of ethanol.
HPLC conditions: Gemini® C18 column (3 x 150 mm, 5 µm); mobile phase:
CH3CN/H2O 0.1% Et3N (v/v, 40/60); flowrate: 0.8 mL/min. TBAOH
tetrabutylammonium hydroxide, DMF = N,N-dimethylformide.
+
=
With radioligand [11C]6 in hand, we then carried out
autoradiography studies in brain sections of Sprague Dawley rats
(Figure 2) and non-human primates (Figure 3) to investigate in
vitro binding specificity towards LRRK2. As shown in Figure 2, in
rat brain sections, under baseline conditions, [11C]6 exhibited
heterogeneous distribution of radioactivity with the highest level
in hippocampus, followed by cerebral cortex and striatum, and the
lowest level in cerebellum, which is consistent with the expression
pattern of LRRK2 in rodents (Figure 2A and 2B). Pretreatment
with PF-06447475, a commercially available LRRK2 inhibitor,
conferred a significant reduction of the bound radioactivity (ca.
50%) in all regional brains, suggesting excellent in vitro binding
specificity of [11C]6 in rat brain towards LRRK2 (Figure 2C and
2D). The high level of in vitro specific binding of [11C]6 to LRRK2
was also demonstrated in non-human primate (NHP, rhesus
monkey) brain sections, with 49% and 47% reduction of
radioactivity in cortex (including all cortical areas, lateral and
medial orbital cortex) and caudate nucleus, respectively, under
blocking conditions (Figure 3).
Figure 3. In vitro autoradiography of [11C]6 in rhesus monkey brain sections. (A)
Brain sections were treated with [11C]6; (B) Brain sections were pre-treated with
PF-06447475 (10 μM), a known LRRK2 inhibitor, followed by [11C]6; (C)
Quantification of the radioactivity of [11C]6 in cortex (including all cortical areas,
lateral and medial orbital cortex) and caudate nucleus under control and
blocking conditions. The data were normalized to % of radioactivity vs control
(n = 3). Asterisks indicate statistical significance. *p < 0.05, and **p ≤0.01.
was evaluated by performing whole-body ex vivo biodistribution
studies in mice at four time points (5, 15, 30 and 60 min) after
intravenous tracer injection. As illustrated in Figure 4 and Table
The uptake, biodistribution and clearance of radioligand [11C]6
3
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S1 in the Supporting Information, radioligand [11C]6 exhibited
limited brain uptake (1.85% ID/g, injected dose per gram of wet
tissue at 5 min), suggesting insufficient BBB permeability in
rodents. High uptake (>5% ID/g) showed up in multiple peripheral
organs including heart, lungs, liver, pancreas, kidneys, small
intestine and stomach, at 5 min, which is in line with the LRRK2
peripheral expression. While the radioactivity in most organs was
rapidly washed out after 5 min, bound signal in small intestine
reached a plateau at 30 min prior to efficient clearance. At 60 min,
high radioactivity was retained in liver, kidneys and small intestine.
These results indicated that radioligand was eliminated through
both urinary and hepatobiliary pathways.
quintet, sext = sextet, sept = setpet, m = multiplet, br = broad
signal, dd = doublet of doublets. Molar activity determinations are
reported at the end of synthesis, unless otherwise stated. The
animal experiments were approved by the Institutional Animal
Care and Use Committee of Massachusetts General Hospital
(OLAW Assurance D16-00361). CD-1 mice (female; 7 weeks, 22-
24 g) were kept on a 12 h light/12 h dark cycle and were allowed
food and water ad libitum.
(3-methoxy-4-nitrophenyl)(morpholino)methanone (2): Under nitrogen
atmosphere, to a 100 mL two-neck flask containing a condenser was
added 3-methoxy-4-nitrobenzoic acid
1
(1 g, 5.0 mmol), 1,2-
dimethoxyethane (DME, 20 mL) and thionyl chloride (SOCl2, 3 mL, 41
mmol). The resulting mixture was heated to 85 oC and stirred at the same
temperature for 6 h until starting material 1 disappeared as monitored by
TLC. Then the reaction mixture was allowed to cool to room temperature
and evaporated to dryness under reduced pressure to give the crude 3-
methoxy-4-nitrobenzoyl chloride, which was directly re-dissolved in dry
tetrahydrofuran (THF, 20 mL). Under nitrogen atmosphere at 0 oC, N,N-
diisopropylethylamine (DIPEA, 1.8 mL, 10 mmol) and morpholine (0.86 mL,
10 mmol) was successively added to the above solution. The resulting
mixture was stirred at 0 oC for 1 h before water (200 mL) was added. After
stirring for another 10 min, the aqueous phase was extracted with ethyl
acetate (20 mL × 5). The organic phases were combined, washed with
brine, dried over anhydrous Na2SO4, and then concentrated under
reduced pressure. The residue was purified by a silica gel chromatography
(PE / EA = 1 / 3) to yield the product as a yellow solid (1.28 g, 96% over
two steps). 1H NMR (500 MHz, DMSO) δ 7.93 (d, J = 8.2 Hz, 1H), 7.37 (s,
1H), 7.12 (d, J = 8.2 Hz, 1H), 3.95 (s, 3H), 3.70 – 3.51 (m, 6H), 3.30 (s,
2H). 13C NMR (101 MHz, CDCl3) δ 167.97, 153.18, 141.09, 140.06, 125.81,
118.37, 112.64, 66.71, 56.73.
Figure 4. Whole-body ex vivo biodistribution studies of [11C]6 in mice at
four different time points (5, 15, 30 and 60 min) post tracer injection. Data
are expressed as % ID/g (mean ± SD; n = 4). Asterisks indicate statistical
significance. * p < 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
(4-amino-3-methoxyphenyl)(morpholino)methanone (3): To a flask
containing 2 (1 g, 3.76 mmol) and acetic acid (12 mL) was added iron
powder (2.73 g, 48.8 mmol, 13 equiv). Under nitrogen atmosphere, the
In this study, we have efficiently synthesized a novel LRRK2
PET probe [11C]6 ([11C]GNE-1023) in good radiochemical yield,
excellent radiochemical purity and high molar activity.
Autoradiography studies demonstrated excellent in vitro specific
binding of [11C]6 to LRRK2 in rat and NHP brain sections. The
whole-body ex vivo bio-distribution studies exhibited limited brain
accumulation, and excretion pathway, specifically urinary and
hepatobiliary elimination for [11C]6 in mice. Although [11C]6
showed limited brain uptake in rodents, given favorate ADME
profile of 6 in MDCK-MDR1 human Pgp transfected cell lines (Papp
A-B, 18.2 x 10-6 cm/s; Efflux ratio, B-A/A-B, 1.2),[21] species
difference in BBB permeability may exist between rodents and
NHPs.[36-40] Further in vivo evaluation in higher species and
medicinal chemistry modification based on [11C]6 scaffold to
improve the BBB permeability is ongoing in our program.
resulting mixture was stirred at room temperature for 3 h until
2
disappeared as monitored by TLC. The reaction mixture was filtered, and
the filtrate was concentrated in vacuo. The residue was purified by a silica
gel chromatography (CH2Cl2 / MeOH = 40 / 1) to afford the desired product
as a white solid (800 mg, 90%). 1H NMR (500 MHz, DMSO) δ 6.85 (s, 1H),
6.80 (dd, J = 8.0, 1.3 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 5.14 (s, 2H), 3.78
(s, 3H), 3.61 – 3.55 (m, 4H), 3.53 – 3.47 (m, 4H). 13C NMR (101 MHz,
CDCl3) δ 171.00 (s), 146.60 (s), 138.65 (s), 123.86 (s), 120.93 (s), 113.22
(s), 110.24 (s), 66.86 (s), 55.48 (s).
2-chloro-N-methyl-5-(trifluoromethyl)pyrimidin-4-amine (5): To
a
solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine 4 (2 g, 9.2 mmol) in
MeOH (35 mL) was successively added triethylamine (Et3N, 1.31 mL, 9
mmol) and methylamine (2.0 M in MeOH, 5 mL, 10 mmol) at 0 oC. The
resulting mixture was allowed to warm to room temperature and stirred at
the same temperature for 12 h until 4 disappeared as monitored by TLC.
The reaction mixture was then evaporated to dryness in vacuo, and the
residue was purified on a silica gel chromatography (PE / EA = 30 / 1) to
give the product as a white solid (780 mg, 40%). 1H NMR (500 MHz,
DMSO) δ 8.37 (s, 1H), 7.92 (s, 1H), 2.89 (d, J = 4.4 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 163.77 (s), 159.71 (s), 154.79 (q, J = 5.1 Hz), 123.66(q, J
= 2.7 Hz), 106.00(q, J = 0.33 Hz) 28.41 (s).
Experiment Sections
The general procedure for experimental section was described
previously[41] with minor modification in this work. All the
chemicals employed in the synthesis were purchased from
commercial vendors and used without further purification. Thin-
layer chromatography (TLC) was conducted with 0.25 mm silica
gel plates (60F254) and visualized by exposure to UV light (254 nm)
or stained with potassium permanganate. Flash column
chromatography was performed using silica gel (particle size
0.040-0.063 mm). Nuclear magnetic resonance (NMR) spectra
were obtained either on a Bruker spectrometer 300, 400 or 500
MHz. Chemical shifts (δ) are reported in ppm, and coupling
constants are reported in Hertz. The multiplicities are abbreviated
as follows: s = singlet, d = doublet, t = triplet, q = quartet, quint =
(3-methoxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimi-din-2-yl)a-
mino)phenyl)(morpholino)methanone (6, GNE-1023): To
a high-
pressure reactor was successively added 3 (380 mg, 1.6 mmol), 5 (400
mg, 1.9 mmol), 2,2,2-trifluoroacetic acid (TFA, 1 mL) and 2-butyl alcohol
(35 mL). The resulting mixture was heated to 185 oC and stirred at the
same temperature for 12 h before cooling to room temperature. The
reaction mixture was then evaporated to dryness in vacuo, and the residue
was purified by a silica gel chromatography (CH2Cl2 / MeOH = 60 / 1) to
deliver the product as a white solid (242 mg, 31%). Melting point = 177 –
4
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178 oC. 1H NMR (300 MHz, DMSO) δ 8.32 (d, J = 8.2 Hz, 1H), 8.19 (s, 1H),
8.08 (s, 1H), 7.23 (d, J = 3.2 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 7.02 (dd, J
= 8.2, 1.7 Hz, 1H), 3.90 (s, 3H), 3.75 – 3.39 (m, 8H), 2.92 (d, J = 3.1 Hz,
3H). 13C NMR (75 MHz, DMSO) δ 169.33, 161.03, 158.89, 154.75 (d, J =
5.3 Hz),148.77, 130.03, 129.93, 125.28 (q, J = 269.5 Hz),.119.98, 119.75,
110.42, 98.61 (q, J = 31.9 Hz), 66.55, 56.48, 28.49.
solution in advance to determine the specificity of radioligand binding. After
incubation, brain sections were rinsed with ice-cold buffer three times for
2 min, dipped in cold distilled water for 10 seconds. The brain sections
were dried with cold air, then placed on imaging plates (BAS-MS2025, GE
Healthcare, NJ, USA) for optimized contact periods. Autoradiograms were
obtained and regions of interest (ROIs) were carefully drawn with the
reference of naked-eye observation. Radioactivity was measured by an
Amersham Typhoon
radioactivity vs control.
5 analyzer system and normalized to % of
(3-hydroxy-4-((4-(methylamino)-5-(trifluoromethyl)pyrimi-din-2-yl)a-
mino)phenyl)(morpholino)methanone (7): To a solution of 6 (200 mg,
0.49 mmol) in dry CH2Cl2 (10 mL) was added boron tribromide (1.0 M in
CH2Cl2, 2 mL, 2 mmol). The resulting mixture was allowed to warm to room
temperature and stirred at the same temperature for 12 h before saturated
aqueous NaHCO3 solution (15 mL) was added. After stirring for an
additional 5 min, the reaction mixture was transferred to a separatory
funnel, and the organic layer was successively separated, washed with
brine, dried over anhydrous Na2SO4, and then evaporated to dryness in
vacuo. The residue was purified by a silica gel chromatography (CH2Cl2 /
MeOH = 30 / 1) to provide the product as a white solid (135 mg, 69%).
Melting point = 270 – 271 oC. 1H NMR (300 MHz, DMSO) δ 10.42 (s, 1H),
8.21 – 8.12 (m, 3H), 7.24 (d, J = 4.2 Hz, 1H), 6.98 – 6.77 (m, 2H), 3.59 (s,
4H), 3.50 (s, 4H), 2.92 (d, J = 4.3 Hz, 3H). 13C NMR (75 MHz, DMSO) δ
169.38, 160.98, 158.88, 154.64 (q, J = 5.6 Hz), 146.85, 129.99, 129.33,
125.27 (q, J = 269.5 Hz), 120.13, 118.82, 114.61, 98.44 (d, J = 31.7 Hz),
66.60, 28.50.
Whole-body ex vivo biodistribution studies: The general procedure for
ex vivo biodistribution studies was described previously[41, 43] with minor
modification in this work. Briefly, a solution of [11C]6 (50 μCi / 100 µL) was
injected into CD-1 mice via tail vein. These mice (each time point n = 4)
were sacrificed at 5, 15, 30 and 60 min post tracer injection. Major organs,
including whole brain, heart, liver, lungs, spleen, kidneys, small intestine
(including contents), muscle, pancreas, stomach and blood were quickly
harvested and weighed. The radioactivity present in these tissues was
measured using a Cobra Model 5002/5003 autogamma counter, and all
radioactivity measurements were automatically decay corrected based on
the half-life of carbon-11. The results are expressed as the percentage of
injected dose per gram of wet tissue (% ID/g).
Statistical Analysis: Statistical analysis was performed by a student’s
two-tailed t-test, and asterisks were used to indicate statistical
significance: * p < 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
Radiosynthesis of [11C]6 ([11C]GNE-1023). The general procedure for
radiolabeling with
[
11C]CH3I was described previously with minor
modification in this work.[42] Briefly, [11C]CH3I was transferred under helium
gas with heating into a reaction vessel containing the phenolic precursor 7
(1.6 mg), NaOH (4 μL, 1.0 M), and anhydrous DMF (300 μL). After the
radioactivity reached a plateau during transfer, the reaction vessel was
warmed to 80 °C and maintained for 5 min. CH3CN/H2O + 0.1% Et3N (v/v,
50/50, 1.5 mL) was added to the reaction mixture, which was then injected
to a semipreparative HPLC system. HPLC purification was completed on
a X-select® C18 column (10 mm i.d. × 250 mm) using a mobile phase of
CH3CN/H2O + 0.1% Et3N (v/v, 50/50) at a flow rate of 5.0 mL/min. The
Acknowledgements
We thank Elijahu Livni (Nuclear Medicine and Molecular Imaging,
Radiology, MGH and Harvard Medical School) for advice in the
radiosynthesis and Professor Thomas J. Brady (Nuclear Medicine
and Molecular Imaging, Radiology, MGH and Harvard Medical
School) for helpful discussion. We also thank Genentech for
providing the material, helpful discussion and support to publish
our preliminary results. Financial support from NIH grants (NIAAA
AA019431 to J.B.D.and NIAAA AA014106 to David P. Friedman)
is gratefully acknowledged.
retention time for
[
11C]6 was 8.7 min. The radioactive fraction
corresponding to the desired product was collected in a sterile flask, diluted
with 30 mL of water, trapped on a Sep-Pak light C18 cartridge. After
washing with 10 mL of water to remove CH3CN residue, the product was
washed out from the cartridge with 1 mL of ethanol, and formulated with
10 mL of PBS-buffered saline. The total synthetic time was ∼42 min from
the end of bombardment. Radiochemical and chemical purity were
measured by analytical HPLC (Gemini® C18 column, 3 mm i.d. × 150 mm,
UV at 254 nm; CH3CN/H2O + 0.1% Et3N (v/v, 40/60) at a flow rate of 0.8
mL/min, retention time: 4.9 min). The identity of product was confirmed by
the co-injection with unlabeled compound 6. Radiochemical yield was 10%
non-decay-corrected based on [11C]CO2 with >99% radiochemical purity,
and the molar activity was greater than 37.0 GBq/μmol (1.0 Ci/μmol).
Conflict of interest
The authors declare no conflict of interest.
Keywords: Parkinson’s disease • leucine-rich repeat kinase 2 •
positron emission tomography • carbon-11 • GNE-1023
Measurement of the lipophilicity: The general procedure for lipophilicity
measurement was described previously[41, 43] with minor modification in
this work. Briefly, the measurement of LogD value was carried out by
mixing [11C]6 (radiochemical purity >99%) with n-octanol (3.0 g) and PBS
(0.1 M, 3.0 g) in a test tube. Both n-octanol and PBS were pre-saturated
with each other prior to use. The tube was first vortexed for 5 min, followed
by centrifugation (~3500-4000 rpm) for an additional 5 min. PBS and n-
octanol (0.60 mL each) were aliquoted, weighted and the radioactivity in
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A novel 11C-labeled LRRK2 PET probe [11C]6 ([11C]GNE-1023) was synthesized in good radiochemical yield, excellent radiochemical
purity and high molar activity. Autoradiography studies demonstrated excellent in vitro specific binding of [11C]6 to LRRK2 in several
species including rat and nonhuman primate brain tissues. The whole-body ex vivo bio-distribution studies exhibited limited brain
accumulation, and excretion pathway, specifically urinary and hepatobiliary elimination for [11C]6 in mice. Nevertheless, further
modification based on [11C]6 scaffold is necessary to improve the BBB permeability.
7
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