Development of a carbon-11 PET radiotracer for imaging TRPC5 in the brain

Article abstract of DOI:10.1039/c9ob00893d

The transient receptor potential channel subfamily member 5 (TRPC5) is a calcium permeable cation channel widely expressed in the brain. Accumulating evidence indicates that it plays a crucial role in psychiatric disorders including depression and anxiety. Positron emission tomography (PET) combined with a TRPC5 specific radioligand may provide a unique tool to investigate the functions of TRPC5 in animal disease models to guide drug development targeting TRPC5. To develop a TRPC5 PET radiotracer, the potent TRPC5 inhibitor HC608 was chosen for C-11 radiosynthesis through the N-demethyl amide precursor 7 reacting with [¹¹C]methyl iodide. Under optimized conditions, [¹¹C]HC608 was achieved with good radiochemical yield (25 ± 5%), high chemical and radiochemical purity (>99%), and high specific activity (204-377 GBq μmol^-1, decay corrected to the end of bombardment, EOB). The in vitro autoradiography study revealed that [¹¹C]HC608 specifically binds to TRPC5. Moreover, initial in vivo evaluation of [¹¹C]HC608 performed in rodents and the microPET study in the brain of non-human primates further demonstrated that [¹¹C]HC608 was able to penetrate the blood brain barrier and sufficiently accumulate in the brain. These results suggest that [¹¹C]HC608 has the potential to be a PET tracer for imaging TRPC5 in vivo.

Full text of DOI:10.1039/c9ob00893d

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Development of a carbon-11 PET radiotracer for
imaging TRPC5 in the brain†
Cite this: DOI: 10.1039/c9ob00893d
Yanbo Yu, a Qianwa Liang,a Hui Liu, a Zonghua Luo, a Hongzheng Hu,b
a
Joel S. Perlmuttera,c and Zhude Tu
*
The transient receptor potential channel subfamily member 5 (TRPC5) is a calcium permeable cation
channel widely expressed in the brain. Accumulating evidence indicates that it plays a crucial role in
psychiatric disorders including depression and anxiety. Positron emission tomography (PET) combined
with a TRPC5 specific radioligand may provide a unique tool to investigate the functions of TRPC5 in
animal disease models to guide drug development targeting TRPC5. To develop a TRPC5 PET radiotracer,
the potent TRPC5 inhibitor HC608 was chosen for C-11 radiosynthesis through the N-demethyl amide
precursor 7 reacting with [11C]methyl iodide. Under optimized conditions, [11C]HC608 was achieved with
good radiochemical yield (25
5%), high chemical and radiochemical purity (>99%), and high specific
activity (204–377 GBq µmol−1, decay corrected to the end of bombardment, EOB). The in vitro auto-
radiography study revealed that [11C]HC608 specifically binds to TRPC5. Moreover, initial in vivo evaluation
of [11C]HC608 performed in rodents and the microPET study in the brain of non-human primates further
demonstrated that [11C]HC608 was able to penetrate the blood brain barrier and sufficiently accumulate
in the brain. These results suggest that [11C]HC608 has the potential to be a PET tracer for imaging
TRPC5 in vivo.
Received 17th April 2019,
Accepted 9th May 2019
DOI: 10.1039/c9ob00893d
rsc.li/obc
or pathological stimuli, TRPC5 channel functions as either
homomeric or heteromeric channel complexes together with
Introduction
Depression is a common cataclysmic psychiatric disorder wide- TRPC1 or TRPC4 regulating intracellular free Ca2+ concen-
spread among world populations and it is one of the leading tration, which subsequently promotes signal transduction,
causes of global medical disability and suicide.1,2 The manage- gene expression and cellular phenotype.6–8 Compelling evi-
ment of depression remains a challenge, due to the lack of dence indicates that TRPC5 plays crucial roles in depression
effective treatments. Usually, 2–4 weeks are required before and other mental disorders.9–11 TRPC5 knock-out mice exhibit
antidepressant medications display efficacy, with approxi- increased exploratory behaviours in both the open field test
mately 30% of patients failing treatment with several different and elevated plus maze (EPM), and have markedly reduced
medications.3 Therefore, new pharmacological targets with symptoms of depression and anxiety.9 Nevertheless, the
faster therapeutic response are urgently needed. Transient precise functions and mechanisms of the TRPC5 channel in
receptor potential channel subfamily member 5 (TRPC5) is a relevant neurological diseases and psychiatric disorders
Ca2+ permeable cation channel that belongs to the transient remain inadequately understood. One of the key challenges is
receptor potential (TRP) superfamily4 and it is widely the lack of highly potent and specific pharmacological probes
expressed in the brain.5 In response to numerous physiological to modulate the channel action in vivo in living animals.
In recent years, several TRPC5 modulators, including activa-
tors such as (−)-englerin A,12 BTD,13 and riluzole14 and inhibi-
tors such as ML204,15 clemizole,16 and AC 190317 have been
reported (Fig. 1). However, these compounds have limitations
aDepartment of Radiology, Washington University School of Medicine, St. Louis,
MO 63110, USA. E-mail: tuz@mir.wustl.edu; Fax: +1 3143628555;
such as their low potency with the IC50 value in the micro-
molar range or poor selectivity for TRCP5 compared to other
TRP channels. More recently, the discovery of 7-(4-chloro-
benzyl)-1-(3-hydroxypropyl)-3-methyl-8-(3-trifluoro-methoxy)-
Tel: +1 3143628487
bDepartment of Anesthesiology, The Center for the Study of Itch,
Washington University School of Medicine in, St. Louis, MO 63110, USA
cDepartment of Neurology, Neuroscience, Physical Therapy and Occupational
Therapy, Washington University School of Medicine, St. Louis, MO 63110, USA
†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectra. See DOI: 10.1039/c9ob00893d
phenoxy)-3,7-dihydro-1H-purine-2,6-dione, HC608,18 was
a
remarkable accomplishment (Fig. 1). Compound HC608, also
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Scheme 1 Synthesis of the precursor 7. Reagents and conditions:
(i) Br2, H2O, 90%; (ii) 1-(bromomethyl)-4-chlorobenzene, K2CO3, DMF,
85%; (iii) (2-(chloromethoxy) ethyl)trimethylsilane, DBU, DMF, 0 °C to
60 °C, 65%; (iv) 2-(3-bromopropoxy)tetrahydro-2H-pyran, K2CO3, DMF,
60 °C, 72%; (v) 3-(trifluoromethoxy)phenol, K2CO3, DMF, 80 °C, 75%;
(vi) HCl (aq), EtOH, reflux, 70%.
Fig. 1 Structures of lead TRPC5 compounds.
known as Pico145 or C31,19 has an IC50 value of 6.2 nM
towards TRPC5, and weaker binding to TRPC4 with an IC50
value of 32.5 nM.20 Importantly, HC608 can distinguish
between closely related channels and has no binding activities
toward other TRP channels including TRPC3 and 6, TRPV
(vanilloid) 1 and 4, TRPA (ankyrin) 1, and TRPM (melastatin)
2 and 8.19 In addition, 7-(4-chlorobenzyl)-8-(3-chlorophenoxy)-1-
(3-hydroxypropyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione,
HC070 (Fig. 1), a structurally close molecule of HC608
produces anxiolytic and antidepressant effects in mice.20
These valuable findings suggest that either [11C]HC608 or
[11C]HC070 could be a promising PET radiotracer targeting
TRPC5. Here we reported our efforts on the synthesis/radio-
synthesis of [11C]HC608 and initial evaluation of [11C]HC608
in rodents and microPET imaging of [11C]HC608 in the brain
of a nonhuman primate to explore the feasibility of [11C]
HC608 to be a PET radiotracer for imaging TRPC5 in vivo.
Although two positions in the structure of 3 could be
N-alkylated, it was found when (2-(chloromethoxy)ethyl)tri-
methylsilane was added into the reaction vial at a lower tem-
perature, intermediate 4 was generated as the major product.
With the 2-(trimethylsilyl)-ethoxymethyl (SEM) group blocked,
the tetrahydropyranyl (THP) ether could be easily added to the
xanthine ring to furnish 5 with a 72% yield. Intermediate 6
was made with a 75% yield via etherification of aryl bromide 5
with 3-(trifluoromethoxy) phenol in DMF in the presence of
K2CO3 at 80 °C. Concentrated hydrochloride solution acted as
a deprotection agent to remove SEM and THP groups simul-
taneously to finalize the synthesis of 7.
To radiosynthesize [11C]HC608 starting from precursor 7,
multiple experimental conditions were tested for generating the
N-[11C]methyl group. Radioactive [11C]CH3I or [11C]methyl tri-
flate ([11C]CH3OTf), solvent, amount of precursor, and different
bases were determined and are summarized in Table 1.
Results and discussion
Table 1 Optimization of the radiosynthesis conditions
Chemistry and radiochemistry
The standard reference compound HC608 and its analogue
HC070 were prepared according to the reported procedure
with necessary modifications.19
HC608 possesses a N-methyl group on a xanthine skeleton
which facilitates utilizing a conventional method to introduce
carbon-11 via N-alkylation of the desmethyl precursor,
7-(4-chlorobenzyl)-1-(3-hydroxypropyl)-8-(3-(trifluoromethoxy)
phenoxy)-3,7-dihydro-1H-purine-2,6-dione (7), reacting with
[11C]triflate or [11C]methyl iodide. As illustrated in Scheme 1,
the synthesis of precursor 7 was achieved by starting with the
Entry Precursor
[
11C] Source
Base
Yield
1
2
3
4
5
6
7, 1.0 mg
7, 1.0 mg
7, 1.0 mg
7, 1.0 mg
7, 1.0 mg
7, 0.6–0.8 mg
[
[
[
[
[
[
11C]CH3I
NaOH (5 M), 2 µL NPa,b
—
11C]CH3OTf
NPa,b
11C]CH3OTf NaOH (5 M), 2 µL NPa,b
11C]CH3I
11C]CH3I
11C]CH3I
NaOH (dry), 5 mg NPa,b
K2CO3 (dry), 5 mg 16%c
K2CO3 (dry), 2 mg 25 5%c,d
commercially procured xanthine 1. Using
a solution of
a No yield, meaning no product was detected by HPLC. b Using DMSO
as the solvent. c Radiochemical yield based on the decay corrected
yield determined by HPLC. d Reaction was performed for more than 10
batches.
bromine to convert compound 1 to 8-bromoxanthine 2, fol-
lowed by N-alkylation on the imidazole ring of the purine
scaffold, intermediate
3 was recovered with high yield.
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Table 2 Biodistribution of [11C]HC608 at 5, 30, and 60 min post injec-
tion in SD rats (n = 4)
50–60 min from the bombardment of [11C]CO2 to the formu-
lation of the injection dose of [11C]HC608 with a good radio-
chemistry yield of 25 5% (decayed to the end of bombardment
(EOB), n > 10), high radiochemical purity (>99%) and high
specific activity (SA = 204–377 GBq µmol−1 (EOB). In the process
of making this radiotracer, it was observed that the injection
dose of [11C]HC608 was sticky and a significant amount of
radioactivity might be retained within the syringe when trans-
ferred into other vials or injected into animals. To avoid exces-
sive product retention in the syringe, 5% polyethylene glycol
(15)-hydroxystearate (Kolliphor® HS15), a potent non-ionic solu-
bilizer and emulsifying agent, was applied for formulating the
injection dose.20
Tracer uptake (%ID per gram)
Tissues
5 min
30 min
60 min
Blood
Heart
Lung
Muscle
Fat
Pancreas
Spleen
Kidney
Thymus
Liver
0.22 0.05
2.06 0.58
1.65 0.44
0.22 0.06
0.14 0.05
1.72 0.38
1.26 0.28
2.59 0.50
0.52 0.16
4.54 1.03
0.51 0.12
0.20 0.01
1.04 0.05
1.13 0.05
0.50 0.02
0.72 0.05
1.49 0.04
0.63 0.03
1.34 0.05
0.63 0.06
3.03 0.51
0.37 0.01
0.16 0.01
0.61 0.13
0.63 0.13
0.36 0.05
0.86 0.23
0.94 0.20
0.37 0.06
0.82 0.16
0.43 0.07
2.22 0.42
0.25 0.03
Brain
Ex vivo biodistribution study of [11C]HC608 in rats
To investigate the kinetics and tissue distribution of
[11C]HC608 in rodents, we performed an ex vivo biodistribution
study using male adult Sprague Dawley (SD) rats. After injec-
tion of the radiotracer, animals were euthanized at 5, 30 and
The optimized conditions include: 0.6–0.8 mg of precursor 7,
300 µL of DMF, 2.0 mg of dry K2CO3, and heating for 5 min at
90 °C (Table 1, entry 6). Under these conditions, it took
Fig. 2
[
11C]HC608 autoradiography of rat brain sections with a radioactive dosage of 0.74, 1.11, and 1.48 MBq per slide respectively under low and
high stringent washing conditions. A. [11C]HC608 autoradiography of rat brain sections under low stringent washing conditions. Tracer dosages were
0.74, 1.11, and 1.48 MBq per slide. B. [11C]HC608 autoradiography of rat brain sections under high stringent washing conditions. C. ARG signals of
sections of three rat brain tissue (n = 3, means SD) under low stringent washing conditions. ARG signal intensity (PSL mm−2) was calculated by
dividing the total signal (PSL) with the total area (mm2) of the section. D. ARG signal of sections of three rat brain tissue (n = 3, means SD) under
high stringent washing conditions. E. The total-to-non-specific-binding ratio is the average signal intensity of three brain sections in each treatment
without blocker over that with blocker (HC070).
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60 min (n = 4 rats per group). The tissue uptake of the radio-
activity was presented as the percentage of injected dose per
gram wet tissue (%ID per gram) in Table 2. Among the
selected tissues, heart, lung, pancreas, kidney and liver had
relatively high uptake (>1.5%ID per gram) at 5 min post injec-
tion. The radioactivity washed out quickly from all tissues
except liver; at 60 min, liver retained 2.22%ID per gram radio-
activity, while for all other tissues the tracer uptake
was <1.0%ID per gram. The initial brain uptake (%ID per
gram) was moderate with 0.51 at 5 min, 0.37 at 30 min, and
0.25 at 60 min. The brain uptake ratio at 5 min versus 60 min
was 2.04, suggesting that [11C]HC608 penetrates the blood
brain barrier and has sufficient accumulation in the rat
brain.
In vitro autoradiography (ARG) and H&E staining study
To check the distribution of [11C]HC608 in the TRPC5-
enriched brain regions of interest, an in vitro autoradiography
study was performed using 20 µm SD rat brain frozen sections.
Brain sections were incubated with [11C]HC608 at different
dosages for 30 min at room temperature. For a blocking study,
HC070, a compound structurally similar to [11C]HC608, with
IC50 values of 9.3 and 46.0 nM for TRPC5 and 4, respectively,
was utilized at 1.0 µM concentration. After 30 min incubation,
sections were washed under low and high stringent washing
conditions.
Fig. 3 The representative sagittal, horizontal, and coronal sections of
rat brains. The sections are showing their ARG signal pattern and their
anatomic structure of H&E staining from the same brain section. BS,
brain stem; Cbm, cerebellum; Ctx, cortex; Hipp, hippocampus; Stri,
striatum; Thal, thalamus; SN, substantia nigra.
ARG signals on the rat brain sections with [11C]HC608 at
2.22 MBq per slide or higher were at saturation levels whether
low or high stringent washing conditions were employed
(data not shown). Tracer dosages at 0.74, 1.11, and 1.48 MBq
per slide gave good ARG signals on rat brain sections as
shown in Fig. 2. A positive ARG signal lighted up essentially
all major regions with strong ARG signals on cortex, hippo- coronal sections are illustrated in Fig. 3. High ARG signals in
campus, midbrain, and brain stem regions. A tracer dose at the cortex, hippocampus, thalamus, mid brain, striatum, and
1.11 MBq per slide provided the best results, while a dose at brain stem regions were observed. These findings recapitulate
1.48 MBq per slide was too high, doses at 0.74 MBq per slide the findings in earlier reports.21–23
yielded poor ratios between ARG signals with and without the
The short half-life of carbon-11 (20.3 min) limits the radio-
blocker regardless of washing conditions. In addition, high activity on the slides during the incubation process for auto-
stringent washing conditions resulted in less tissue coming radiography. To make sure sufficient signal was generated
off the sections and
a lower background ARG signal. when performing the in vitro autoradiography using
Consequently, we set 1.11 MBq per slide under high stringent [11C]HC608 for brain slides, we started with a high tracer dose
washing conditions as optimized parameters for the ARG at 6.29 MBq per slide. Optimal outcomes were achieved with a
study. Remarkably, under this condition, the presence of the radioactive dose of the 11C-tracer at 1.11 MBq; no more than
blocking agent HC070 (1 µM) significantly reduced the in vitro 1.48 MBq per slide is needed. The high lipophilicity of
ARG signal, and the ratio of the average signal intensity for the [11C]HC608 is one of our concerns for autoradiography studies
baseline over the blocking slide was 2.44 : 1 (∼60% reduce). since brain tissue is rich in lipids, and lipophilic [11C]HC608
Overall, our ARG studies suggest: (a) TRPC4/5 TRPC5 channels may require stringent washing conditions, such as washing
on the frozen rat brain tissue remain biologically active, (b) [11C] buffers with a high concentration of alcohol to reduce the non-
HC608 retains strong binding activities to TRPC5 channels on specific background of ARG signals. However, the stringent
rat brain sections, (c) HC070 significantly reduces [11C]HC608 washing buffers may also disrupt the protein structure confor-
uptake, demonstrating the specific binding of [11C]HC608 to mation and components of TRPC channels, and suppress the
TRPC5 in the brain slides.
ARG signal of specific binding on rat brain sections. Our opti-
After the ARG experiment, rat brain sections were co- mized in vitro ARG protocol indicates that a washing buffer
stained with Haemotoxylin and Eosin (H&E) to show their ana- with alcohol up to 30% is compatible for detecting the
tomic structure. The matching alignments between the ARG binding of [11C]HC608 to TRPC5 channels on rat brain
signal and anatomic structures on sagittal, horizontal, and sections.
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Experimental
Materials
All the starting materials, and solvents were purchased com-
mercially and used as received, unless otherwise stated.
Reactions were monitored by thin-layer chromatography (TLC)
using silica gel 60 F254 (EMD Chemicals Inc, Billerica, MA)
Flash column chromatography was conducted using
230–400 mesh silica gel (SiliCycle Inc, Quebec, Canada).
Melting points were determined using MEL-TEMP 3.0
apparatus without correction. 1H NMR and 13C NMR spectra
were recorded at 400 MHz on a Varian spectrometer with
CDCl3 as the solvent and tetramethylsilane (TMS) as the
internal standard. All chemical shift values are reported
in parts per million (ppm) and were calibrated using a
residual undeuterated solvent as an internal reference (CDCl3:
δ 7.26 ppm). Coupling constants (J) are reported in Hertz (Hz).
The following abbreviations are used to describe multiplicities
– s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
and br. (broad). High resolution mass spectra (HRMS, m/z)
were recorded by using a Bruker MaXis 4G Q-TOF mass
spectrometer with an electrospray ionization source.
Fig. 4 Summed transverse and coronal PET images (upper panel) and
time-activity curve (lower panel) of [11C]HC608 for the microPET study
in the whole brain of a male adult cynomolgus macaque. SUV: standar-
dized uptake value.
All animal experiments were conducted under Washington
University’s Institutional Animal Care and Use Committee
(IACUC)-approved protocols in accordance with the US
National Research Council’s Guide for the Care and Use of
Laboratory Animals.
Non-human primate (NHP) microPET study
To further investigate the ability to cross the blood brain
barrier and the in vivo kinetics of [11C]HC608 in the NHP
brain, a microPET study was conducted on a male adult
cynomolgus macaque. As displayed in Fig. 4, the brain
standardized uptake value (SUV) reached a maximum (∼1.1)
at 6–10 min post tracer injection, followed by a fast washout
from the NHP brain. The uptake (SUV) ratio of [11C]HC608
at 8 min versus 120 min reached 2.75-fold. The microPET
data suggest that [11C]HC608 crosses the blood brain barrier
and possesses fast washout kinetics in the NHP brain
although the brain uptake is relatively low as a brain PET
radiotracer.
Chemistry
The standard compound HC608 and blocking agent HC070
were synthesized via 4 steps according to a reported procedure
with modification.19 Briefly, 1-(bromomethyl)-4-chorobenzene
(1.25 g, 6.1 mmol) was added to a suspension of 8-bromo-
3-methyl-1H-purine-2,6(3H,7H)-dione (1.5 g, 6.1 mmol) and
K2CO3 (0.85 g, 6.1 mmol) in DMF (10 mL). The mixture was
stirred at rt for 1.5 hours before another equivalent of K2CO3
(0.85 g, 6.1 mmol) and 2-(3-bromopropoxy)tetrahydro-2H-pyran
(1.1 mL, 6.4 mmol) were added, and the mixture was stirred
overnight at 60 °C. After cooling, the mixture was poured into
water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The
combined organic phases were dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo to yield a white solid, which
Conclusions
In conclusion, we have successfully synthesized the first was then suspended in EtOH (35 mL). Concentrated aqueous
TRPC5 PET radioligand, [11C]HC608, with good radiochemical HCl (5 mL) was added. The mixture was heated at reflux for
yield, high chemical and radiochemical purity, and high about 8 h and concentrated to dryness in vacuo. Then the
specific activity. Our initial ex vivo biodistribution and in vitro residue was diluted with ethyl acetate (50 mL) and washed with
autoradiography studies in rodents and microPET study in the water (20 mL) and brine. The organic phase was dried over an-
NHP brain suggest that [11C]HC608 specifically binds to hydrous Na2SO4 and concentrated to give a yellow oil. In the last
TRPC5 in the rat brain sections and penetrates the blood brain step, the product obtained above was mixed with substitute
barrier and accumulates in the brain of rats and a NHP. phenol (6.1 mmol) and K2CO3 (1.7 g, 12.2 mmol) in DMF
Together, these findings indicate that [11C]HC608 has a (10 mL) and stirred at 80 °C overnight. After cooling to rt, the
great potential to be a PET radiotracer for in vivo quantification reaction solution was diluted with water (30 mL) and extracted
of TRPC5 expression in the brain. Further investigation of with ethyl acetate (3 × 50 mL). The combined organic layers were
[11C]HC608 in animal models of neurological diseases will washed with brine, dried with Na2SO4 and concentrated
advance our understanding of the functions of TRPC5 in the in vacuo. The residue was purified by flash column chromato-
brain.
graphy (hexane/ethyl acetate, 2/1, v/v) to provide the final product.
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7-(4-Chlorobenzyl)-1-(3-hydroxypropyl)-3-methyl-8-(3-(tri- mixture was quenched with water and extracted with ethyl
fluoromethoxy)phenoxy)-3,7-dihydro-1H-purine-2,6-dione acetate (3 × 100 mL). The combined organic phases were
(HC608). Yield 20% over 5 steps; white solid; Mp 120–121 °C. washed with brine, dried over anhydrous Na2SO4, filtered, and
1H NMR (400 MHz, CDCl3) δ 7.47 (t, J = 8.1 Hz, 1H), 7.42 (d, J = concentrated in vacuo. The residue was purified by flash
8.0 Hz, 2H), 7.34–7.31 (m, 2H), 7.25 (d, J = 8.6 Hz, 2H), 7.17 (d, column chromatography (hexane/ethyl acetate, 3 : 1 to 1 : 1, v/v)
J = 8.0 Hz, 1H), 5.44 (s, 2H), 4.22–4.20 (m, 2H), 3.58–3.54 (m, to provide the product as a white solid (892 mg, 65% yield).
3H), 3.45 (s, 3H), 1.93–1.92 (m, 2H). 13C NMR (101 MHz, Mp 148–149 °C. 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H),
CDCl3) δ 155.19, 153.47, 152.76, 151.52, 149.66, 146.20, 134.45, 7.37–7.30 (m, 4H), 5.47 (s, 4H), 3.72 (d, J = 8.4 Hz, 2H), 0.97
134.11, 130.60, 129.76, 129.10, 120.29 (q, J = 258.4 Hz), 118.21, (d, J = 8.5 Hz, 2H), 0.02 (s, 9H). 13C NMR (101 MHz, CDCl3)
117.87, 112.98, 102.91, 58.51, 46.57, 37.73, 30.85, 29.90. HRMS δ 153.72, 150.52, 149.84, 134.86, 133.23, 129.76, 129.30,
(ESI) calcd for C23H21ClF3N4O5 [M + H]+ 525.1147, found: 128.59, 109.19, 71.60, 67.75, 49.88, 18.13, −1.31. HRMS
525.1150.
(ESI) calcd for C18H23BrClN4O3Si [M + H]+ 485.0406, found:
7-(4-Chlorobenzyl)-8-(3-chlorophenoxy)-1-(3-hydroxypropyl)- 485.0400.
3-methyl-3,7-dihydro-1H-purine-2,6-dione (HC-070). Yield
8-Bromobromo-7-(4-chlorobenzyl)-1-(3-((tetrahydro-2H-pyran-
18% over 5 steps; white solid; Mp 131–132 °C. 1H NMR 2-yl)oxy)propyl)-3-((2-(trimethylsilyl)ethoxy)methyl)-3,7-dihydro-
(400 MHz, CDCl3) δ 7.34–7.19 (m, 7H), 7.10 (d, J = 7.9 Hz, 1H), 1H-purine-2,6-dione (5). Compound 4 (895 mg, 1.85 mmol),
5.34 (s, 2H), 4.13–4.11 (m, 2H), 3.46–3.44 (m, 3H), 3.37 (s, 3H), 2-(3-bromopropoxy)tetrahydro-2H-pyran (453 mg, 2.04 mmol,
1.84–1.82 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 155.32, 1.1 equiv.), and K2CO3 (511 mg, 3.7 mmol, 2 equiv.) were
153.45, 153.07, 151.65, 146.39, 135.19, 134.56, 134.24, 130.66, mixed in DMF (10 mL) and heated at 60 °C overnight. At com-
129.91, 129.23, 126.38, 120.36, 118.00, 102.99, 58.58, 46.67, pletion, the reaction mixture was partitioned between water
37.81, 30.95, 30.12. HRMS (ESI) calcd for C22H21Cl2N4O4 (50 mL) and ethyl acetate (50 mL). The organic phase was sep-
[M + H]+ 475.0940, found: 475.0934.
arated, and the aqueous phase was washed with ethyl acetate
8-Bromo-3,7-dihydro-1H-purine-2,6-dione (2). Under a N2 (2 × 50 mL). The combined organic solution was washed with
atmosphere, xanthine 1 (10.0 g, 66 mmol) and distilled water brine, dried over anhydrous Na2SO4, filtered and concentrated.
(65 mL) were added sequentially to a 300 mL sealed tube. The crude was purified by flash column chromatography
Then Br2 (5.0 mL, 99 mmol, 1.5 equiv.) was added slowly with (hexane/ethyl acetate, 4 : 1 to 1 : 1, v/v) to provide the product
stirring. The sealed tube was capped and heated to 100 °C for as a colorless oil (834 mg, 72% yield). 1H NMR (400 MHz,
4 hours. After cooling to room temperature, the reaction CDCl3) δ 7.35 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.5 Hz, 2H),
mixture was filtered and the solid participate was washed with 5.45–5.55 (m, 4H), 4.57 (s, 1H), 4.24–4.05 (m, 2H), 3.85–3.73
water and Et2O. The product was dried in a vacuum to give (m, 2H), 3.78–3.69 (m, 2H), 3.53–3.41 (m, 2H), 2.02–1.91 (m,
2 (13.66 g, 90% yield) as a white solid. 1H (400 MHz, DMSO-d6) 2H), 1.86–1.72 (m, 1H), 1.68–1.57 (m, 1H), 1.55–1.40 (m, 4H),
δ 11.65 (s, 1H), 10.92 (s, 1H). 13C NMR (101 MHz, DMSO-d6) 0.99 (t, J = 8.1 Hz, 2H), 0.00 (s, 9H). 13C NMR (101 MHz,
δ 154.49, 151.01, 148.77, 124.22, 109.42. HRMS (ESI) calcd for CDCl3) δ 170.61, 153.94, 150.65, 147.58, 134.14, 133.31, 129.24,
C5H4BrN4O2 [M + H]+ 230.9512, found: 230.9519.
128.78, 127.55, 108.58, 98.32, 71.73, 67.15, 65.04, 61.74,
8-Bromo-7-(4-chlorobenzyl)-3,7-dihydro-1H-purine-2,6-dione (3). 60.04, 49.18, 39.10, 30.37, 27.89, 25.28, 20.76, 19.17, 17.68,
Compound 2 (3.45 g, 15 mmol) and K2CO3 (6.21 g, 45 mmol, 14.01, −1.64. HRMS (ESI) calcd for C26H37BrClN4O5Si [M + H]+
3
equiv.) were dissolved in DMF (30 mL), and 627.1400, found: 627.1401.
then 1-(bromomethyl)-4-chlorobenzene (3.37 g, 16.5 mmol, 7-(4-Chlorobenzyl)-1-(3-((tetrahydro-2H-pyran-2-yl)oxy)-propyl)-
1.1 equiv.) was added dropwise. The reaction mixture was 8-(3-(trifluoromethoxy)phenoxy)-3-((2-(trimethyl-silyl)ethoxy)-
heated for another 2 hours at 45 °C and quenched by the methyl)-3,7-dihydro-1H-purine-2,6-dione (6). Compound
5
addition of water, extracted with ethyl acetate (3 × 100 mL). (834 mg, 1.33 mmol), 3-(trifluoro-methoxy)phenol (284 mg,
The combined organic solution was washed with brine, dried 1.66 mmol, 1.2 equiv.) and K2CO3 (367 mg, 2.66 mmol,
over anhydrous Na2SO4, filtered, and concentrated under 2 equiv.) were mixed in DMF (10 mL) and heated at 80 °C over-
reduced pressure to afford 3 as a white solid (4.51 g, 85% night. After cooling to rt, the resulting solution was quenched
yield). Mp 300–301 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.40 (d, with water and extracted with ethyl acetate (3 × 100 mL). The
J = 8.0 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 5.38 (s, 2H). 13C NMR combined organic phases were washed with brine, dried over
(101 MHz, DMSO-d6) δ 156.29, 135.86, 132.25, 129.12, 128.61, anhydrous Na2SO4, filtered, and concentrated under reduced
127.03, 108.22, 48.19. HRMS (ESI) calcd for C12H8O2N4BrClNa pressure. The residue was purified by flash column chromato-
[M + Na]+ 376.9411, found: 376.9421.
graphy (hexane/ethyl acetate, 4 : 1 to 3 : 1, v/v) to yield a colour-
8-Bromo-7-(4-chlorobenzyl)-3-((2-trimethylsilyl)ethoxy)- less oil product (722 mg, 75% yield). 1H NMR (400 MHz,
methyl)-3,7-dihydro-1H-purine-2,6-dione (4). To a stirred solu- CDCl3) δ 7.52–7.47 (m, 3H), 7.39–7.33 (m, 4H), 7.22–7.20 (m,
tion of 3 (1.01 g, 2.84 mmol), DBU (432 mg, 2.84 mmol, 1H), 5.51–5.46 (m, 5H), 4.67–4.66 (m, 1H), 4.28–4.13 (m, 3H),
1.0 equiv.) in DMF (20 mL) was added 2-(trimethyl-silyl)ethoxy- 3.93–3.91 (m, 2H), 3.77–3.72 (m, 2H), 3.57–3.55 (m, 2H),
methylethoxymethyl chloride (476 mg, 2.84 mmol, 1.0 equiv.) 2.09–2.06 (m, 3H), 1.86–1.84 (m, 1H), 1.73–1.71 (m, 1H),
at 0 °C. Then the mixture was warmed to rt and heated at 1.34–1.28 (m, 1H), 0.99 (t, J = 7.6 Hz, 2H), 0.00 (s, 9H).
60 °C until TLC demonstrated the reaction was complete. The 13C NMR (101 MHz, CDCl3) δ 154.63, 153.58, 152.23, 151.05,
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149.53, 145.14, 134.22, 130.44, 129.67, 128.96, 120.24 (q, J = (0.6–0.8 mg) and K2CO3 (2.0 mg) in DMF (0.3 mL) at room
258.2 Hz).117.92, 117.74, 112.77, 102.99, 98.47, 71.94, 67.28, temperature. Right after the trap of radioactivity reached a
65.23, 61.85, 60.17, 46.38, 38.99, 30.51, 28.13, 25.40, 19.27, plateau, the sealed reaction vessel was heated for 5 min using
17.75, 14.05, −1.67, −1.70. HRMS (ESI) calcd for an oil bath at 90 °C. After cooling, the reaction mixture was
C33H40ClF3N4O7SiNa [M + Na]+ 747.2199, found: 747.2198.
quenched with 1.7 mL of HPLC mobile phase consisting of
7-(4-Chlorobenzyl)-1-(3-hydroxypropyl)-8-(3-(trifluoro-methoxy)- 62% acetonitrile and 38% of 0.1 M ammonium formate (v/v,
phenoxy)-3,7-dihydro-1H-purine-2,6-dione (7). To a stirred solu- pH 4.5). The diluted reaction mixture was loaded onto a
tion of compound 6 (608 mg, 0.84 mmol) in EtOH (10 mL) was reverse phase semi-prepared C-18 Agilent column (Zorbax
added concentrated hydrochloric acid (1.3 mL) dropwise. Then SB-C18, 9.2 mm ID × 250, 5 µm) using the abovementioned
the mixture was heated under reflux and monitored by TLC HPLC mobile phase at a flow rate of 4 mL min−1 and a UV
until complete consumption of the starting material. After detector wavelength of 254 nm. The radioactive product was
about 8 hours, the reaction solution was cooled and quenched collected from 15 to 16 min into a vial that contained 50 mL of
with 10 mL NaHCO3 (sat.), diluted with water (30 mL) and Milli-Q water. The diluted radioactive solution was passed
ethyl acetate (50 mL). The organic phase was separated, and through
a plus short C-18 Sep-Pak cartridge (Part No.
the aqueous phase was washed with ethyl acetate (2 × 50 mL). WAT020515, Waters Corporation, Milford, MA). After using
The combined organic solution was washed with brine, dried 20 mL of Milli-Q water to rinse the C-18 cartridge, the radio-
over anhydrous Na2SO4, filtered and concentrated to dryness active product was eluted into a dose vial using 1.0 mL absol-
in vacuo. The crude was purified by flash column chromato- ute ethanol, and then 9.0 mL solution of 5% Kolliphor HS15
graphy (hexane/ethyl acetate, 1 : 2 to 1 : 3, v/v) to provide the (Sigma-Aldrich) and 85% saline (v/v) was added into the dose
product as a white solid (300 mg, 70% yield). Mp 200–201 °C. vial to formulate an 10% ethanol 10 mL solution prepared for
1H NMR (400 MHz, DMSO-d6) δ 11.92 (s, 1H), 7.59 (t, J = intravenous (IV) injection into animals.
8.3 Hz, 1H), 7.48–7.37 (m, 6H), 7.32–7.30 (m, 1H), 5.40 (s, 2H),
An aliquot of the dose sample (20 µL) was delivered to an
4.46 (t, J = 5.2 Hz, 1H), 3.97–3.79 (m, 2H), 3.45–3.41 (m, 2H), analytic HPLC system for authentication by co-injection with
1.88–1.38 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 154.70, the cold standard compound HC608 and determination of the
153.74, 153.02, 150.58, 148.73 (q, J = 1.8 Hz), 144.66, 135.37, chemical and radiochemical purity, and specific activity. The
132.62, 131.36, 129.59, 128.76, 119.96 (q, J = 257.2 Hz), 119.51, quality control HPLC system consisted of an analytical reverse
118.42, 113.65, 102.58, 58.84, 45.74, 37.52, 31.11. HRMS (ESI) phase HPLC column (Agilent Zorbax SB-C18, 4.6 mm × 250),
calcd for C22H18ClF3N4O5Na [M + Na]+ 533.0810, found: UV wavelength of 254 nm, mobile phase of CH3CN/0.1 M
533.0809.
HCOONH4 (v/v, 82/18, pH = 4.5) with the flow rate of
1.0 mL min−1. Under this condition, retention time TR
5.4 min. It confirmed that the radioactive product was
=
Radiochemistry
[11C]CO2 was produced by the proton bombardment of nitro- [11C]HC608 with high specific activity (204–377 GBq µmol−1
,
gen-14 gas via the 14N (p, α)11C nuclear reaction using the JSW decay corrected to EOB) and >99% chemical and radio-
BC-16/8 cyclotron of Washington University Medical School in chemical purity.
Saint Louis. The [11C]methyl iodide ([11C]CH3I) was generated
Ex vivo biodistribution study in rats
by the reduction of [11C]CO2 using a nickel catalyst under an
atmosphere of H2 at 360 °C, followed by iodination using iodine A solution of [11C]HC608 (∼3.7 MBq per 100 µL, in 10%
at 690 °C to prepare [11C]CH3I. Also, the [11C]CH3I generated ethanol, 5% Kolliphor HS15, 85% saline) was injected via the
was run through the silver triflate (AgOTf) column to produce tail vein into Sprague Dawley (SD) rats (male; 7 weeks old;
[11C]CH3OTf at 228 °C. The resulting [11C]CH3I or [11C]CH3OTf 200–220 g). Post injection of the radioactivity, rats were eutha-
was transferred via gas tubing to the reaction vessel in a hot cell. nized under anaesthesia at 5, 30, and 60 min post-injection
For radiolabelling reaction condition screening, precursor 7 (n = 4 per group). Tissues of interest (blood, heart, lung,
(0.6–1.0 mg) was dissolved in a solvent (0.3 mL), followed by muscle, fat, pancreas, spleen, kidney, liver, thymus, and whole
base addition, then the [11C] reagent was bubbled into the brain) were collected, weighed and counted on an automated
solution until the activity peaked. The resulting solution was Beckman Gamma counter. The uptake of each organ was cal-
then heated at 90 °C for 5 minutes during which the vial was culated and expressed as a percentage of the injection dose
shaken occasionally using a long clamp. Then the oil bath was per gram of wet tissue (%ID per gram).
removed and the mixture was cooled and quenched with
1.7 mL HPLC mobile phase. After that, the resulting solution
In vitro autoradiography and H & E staining study
was injected onto the semi preparative HPLC column and the Frozen sections (20 micron) were prepared from adult Sprague
desired radioactivity fraction was collected, diluted with 50 mL Dawley (SD) rat brains (male; 7 weeks old; 200–220 g). The
water, and loaded onto a C18 Sep-Pak cartridge. The desired slides were incubated with [11C]HC608 (0.74–1.48 MBq per
[11C]HC608 solution was eluted out with 1.0 mL absolute slide) at room temperature for 30 min with or without the pres-
ethanol elution.
ence of 1 µM of HC070. After incubation, the brain slides were
Optimal conditions are given in Table 1, entry 6. [11C]CH3I washed for 2 min each in the following buffers sequentially:
was bubbled for 2–3 min into a solution of precursor 7 1× PBS, 10% EtOH in 1× PBS, 30% EtOH in 1× PBS, and
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1× PBS (For low stringent washing conditions: 1× PBS, 5% also grateful to John Hood, Emily Williams, and Darryl Craig
EtOH in 1× PBS, 15% EtOH in 1× PBS and 1× PBS). Then the for their assistance with the nonhuman primate microPET
rat brain sections were exposed on the Storage Phosphor studies. We appreciate Drs. Jiwei Gu, Lin Qiu, Tangadanchu
Screen in an imaging cassette at −20 °C in the dark for 3 h. Vijai Kumar Reddy and Joshi Sumit’s help in the animal study.
The distribution of radioactivity was visualized with a Fuji Bio-
Imaging Analyzer FLA-7000 (Fuji Photo Film, Tokyo, Japan).
Multi Gauge v3.0 software (Fuji Photo Film Co., Tokyo, Japan)
was used to quantify the signal (photo-stimulated lumine-
Notes and references
scence, PSL) from the brain sections. Data were background
corrected and expressed as photo-stimulated luminescence
signals per square millimeter (PSL mm−2).
1 R. Machado-Vieira, I. D. Henter and C. A. Zarate Jr., Prog.
Neurobiol., 2017, 152, 21–37.
2 A. F. Carvalho, M. S. Sharma, A. R. Brunoni, E. Vieta and
G. A. Fava, Psychother. Psychosom., 2016, 85, 270–288.
3 A. J. Rush, M. H. Trivedi, S. R. Wisniewski,
A. A. Nierenberg, J. W. Stewart, D. Warden, G. Niederehe,
M. E. Thase, P. W. Lavori and B. D. Lebowitz,
Am. J. Psychiatry, 2006, 163, 1905–1917.
4 N. Damann, T. Voets and B. Nilius, Curr. Biol., 2008, 18,
R880–R889.
5 M. A. Fowler, K. Sidiropoulou, E. D. Ozkan, C. W. Phillips
and D. C. Cooper, PLoS One, 2007, 2, e573.
6 A. Greka, B. Navarro, E. Oancea, A. Duggan and
D. E. Clapham, Nat. Neurosci., 2003, 6, 837–845.
7 I. Jemal, S. Soriano, A. L. Conte, C. Morenilla and
A. Gomis, Pflugers Arch., 2014, 466, 1635–1646.
8 B. Shen, C.-O. Wong, O.-C. Lau, T. Woo, S. Bai, Y. Huang
and X. Yao, PLoS One, 2015, 10, e0122227.
After autoradiography, the brain sections were air dried for
several minutes before being stained with filtered 0.1%
Mayers Hematoxylin (Sigma; MHS-16) for 4 minutes in a
50 mL conical tube. Subsequently, the sections were rinsed in
cool running double-distilled water for 5 minutes in a Coplin
jar. Then the sections were dipped in 0.5% Eosin (1.5 g dis-
solved in 300 mL 95% EtOH) 12 times and distilled water
until the eosin stops streaking. Next, sections were dipped in
50% and 70% EtOH 10 times separately before being equili-
brated in 95% and 100% EtOH for 30 seconds and 1 minute
respectively. Finally, coverslips were cleaned with kimwipes
and mounted with Cytoseal XYL (Stephens Scientific; cat#
8312-4) before scanning.
Non-human primate (NHP) microPET study
9 A. Riccio, Y. Li, J. Moon, K.-S. Kim, K. S. Smith,
U. Rudolph, S. Gapon, G. L. Yao, E. Tsvetkov and
S. J. Rodig, Cell, 2009, 137, 761–772.
10 K. Sossey-Alaoui, J. A. Lyon, L. Jones, F. E. Abidi,
A. J. Hartung, B. Hane, C. E. Schwartz, R. E. Stevenson and
A. K. Srivastava, Genomics, 1999, 60, 330–340.
11 S. V. Puram, A. Riccio, S. Koirala, Y. Ikeuchi, A. H. Kim,
G. Corfas and A. Bonni, Genes Dev., 2011, 25, 2659–2673.
12 Y. Akbulut, H. J. Gaunt, K. Muraki, M. J. Ludlow,
M. S. Amer, A. Bruns, N. S. Vasudev, L. Radtke, M. Willot
and S. Hahn, Angew. Chem., Int. Ed., 2015, 54, 3787–
3791.
13 H. Beckmann, J. Richter, K. Hill, N. Urban, H. Lemoine
and M. Schaefer, Cell Calcium, 2017, 66, 10–18.
14 J. M. Richter, M. Schaefer and K. Hill, Br. J. Pharmacol.,
2014, 171, 158–170.
A male adult macaque (7.6 kg) was studied with a microPET
Focus 220 scanner (Concorde/CTI/Siemens Microsystems,
Knoxville, TN). The animal was scanned under anesthesia
(induced with ketamine and secretions reduced with glycopyr-
rolate) with inhalation of 1–2% isoflurane in 40% N2O and
60% O2. A 2 h dynamic scan was acquired after a bolus injec-
tion 4.0 mL of 310.8 MBq of [11C]HC608 via a venous catheter.
For quantitative analyses, PET images were co-registered with
MRI images. The three-dimensional region of interest (the
global brain) was identified on the MRI and transformed to
the reconstructed PET images to obtain time-activity curves.
Activity measures were standardized to body weight and the
dose of radioactivity injected to yield SUV.
Conflicts of interest
15 M. Miller, J. Shi, Y. Zhu, M. Kustov, J.-B. Tian, A. Stevens,
M. Wu, J. Xu, S. Long and P. Yang, J. Biol. Chem., 2011, 286,
33436–33446.
There are no conflicts to declare.
16 J. M. Richter, M. Schaefer and K. Hill, Mol. Pharmacol.,
2014, 86, 514–521.
17 Y. Zhou, P. Castonguay, E.-H. Sidhom, A. R. Clark,
M. Dvela-Levitt, S. Kim, J. Sieber, N. Wieder, J. Y. Jung and
S. Andreeva, Science, 2017, 358, 1332–1336.
Acknowledgements
This work was supported by the USA National Institutes of
Health (NIH) through the National Institute of Neurological
Disorders and Stroke, the National Institute on Aging 18 B. Chenard and R. Gallaschun, Xanthines and methods of
[NS075527 and NS103988], the American Parkinson Disease use thereof, Global patent, WO2014/143799A2, 2014.
Association (APDA), the Greater St. Louis Chapter of the APDA 19 H. N. Rubaiy, M. J. Ludlow, M. Henrot, H. J. Gaunt,
and Barnes Jewish Hospital Foundation. We would like to
thank William H. Margenau of Washington University
Cyclotron Facilities for 11C radioisotope production. We are
K. Miteva, S. Y. Cheung, Y. Tanahashi, N. Hamzah,
K. E. Musialowski and N. M. Blythe, J. Biol. Chem., 2017,
292, 8158–8173.
Org. Biomol. Chem.
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Paper
20 S. Just, B. L. Chenard, A. Ceci, T. Strassmaier, 22 Y. H. Chung, H. S. Ahn, D. Kim, D. H. Shin, S. S. Kim,
J. A. Chong, N. T. Blair, R. J. Gallaschun, D. del Camino,
S. Cantin and M. D’Amours, PLoS One, 2018, 13,
e0191225.
K. Y. Kim, W. B. Lee and C. I. Cha, Brain Res., 2006, 1085,
132–137.
23 A. Riccio, A. D. Medhurst, C. Mattei, R. E. Kelsell,
A. R. Calver, A. D. Randall, C. D. Benham and
M. N. Pangalos, Mol. Brain Res., 2002, 109, 95–104.
21 C. Strübing, G. Krapivinsky, L. Krapivinsky and
D. E. Clapham, Neuron, 2001, 29, 645–655.
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