Synthesis and initial preclinical evaluation of the P2X7 receptor antagonist [11C]A-740003 as a novel tracer of neuroinflammation

Article abstract of DOI:10.1002/jlcr.3206

Neuroinflammation, in particular activation of microglia, is thought to play an important role in the progression of neurodegenerative diseases. In activated microglia, the purinergic P2X₇ receptor is upregulated. A-740003, a highly affine and

Full text of DOI:10.1002/jlcr.3206

Research Article
Received 24 February 2014,
Revised 9 April 2014,
Accepted 11 April 2014
Published online 3 July 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3206
Synthesis and initial preclinical evaluation of
11
the P2X7 receptor antagonist [ C]A-740003 as
a novel tracer of neuroinflammation
a
a
a,b
Bieneke Janssen, Danielle J. Vugts, Uta Funke, Arnold Spaans,a,b
Robert C. Schuit,a Esther Kooijman,a Marissa Rongen,a Lars R. Perk,b
Adriaan A. Lammertsma,a and Albert D. Windhorsta
*
Neuroinflammation, in particular activation of microglia, is thought to play an important role in the progression of
neurodegenerative diseases. In activated microglia, the purinergic P2X7 receptor is upregulated. A-740003, a highly affine
and selective P2X7 receptor antagonist, is a promising candidate for the development of a radiotracer for imaging of
neuroinflammation by positron emission tomography. For this purpose, [11C]A-740003 was synthesised and evaluated
in vivo with respect to both tracer metabolism and biodistribution. In plasma, a moderate metabolic rate was seen. In
healthy rat brain, only marginal uptake of [11C]A-740003 was observed and, therefore, metabolites in brain could not be
determined. Whether the minimal brain uptake is due to the low expression levels of the P2X7 receptor in healthy brain
or to limited transport across the blood–brain barrier has yet to be elucidated.
Keywords: radiolabelling; carbon-11; A-740003; P2X7 receptor; neuroinflammation; positron emission tomography
response. Upon binding of ATP to the receptor, microglia are
activated and proliferation of microglia is stimulated. Through
the pore that is then formed, chemokines are released to
Introduction
Neuroinflammation is thought to play a key role in the
progression of several pathologies of the central nervous system
recruit other microglia. Release of proinflammatory cytokines
(CNS), including Alzheimer’s disease, Parkinson’s disease,
(e.g. interleukin-1β (IL-1β) and tumour necrosis factor α (TNFα))
multiple sclerosis and amyotrophic lateral sclerosis.1–3 Microglia,
through the pore leads to further microglial activation.
Furthermore, IL-1β and TNFα are shown to promote neuronal
damage, and subsequent apoptosis leads to increased ATP
release. Continued presence of ATP and an increasing level of
as resident macrophages of the CNS, are involved in the
neuroinflammatory response.2,4 Microglia switch from a resting
to an activated state in response to, for example, tissue damage,
pathogen invasion and protein aggregates. Next to their
involvement in the immune response to clear pathogens and
proinflammatory cytokines maintain the cycle of degeneration
and inflammation.2 According to this model, a P2X7 receptor
promote tissue repair, excessive activity of microglia may also
antagonist might halt the inflammatory cascade and thus further
contribute to the progression of CNS pathologies.2,5 Activated
progression of neurodegeneration. Because of its involvement in
microglia have been found in affected brain areas of various
the process, the P2X7 receptor might be an interesting and novel
pathologies and, therefore, they may be a viable diagnostic
target for positron emission tomography (PET) imaging of
neuroinflammation, where an ideal PET ligand is still missing.
marker for disease onset or progression.5,6 Following their
activation, microglia alter the expression of, amongst others, cell
To date, a variety of P2X7 receptor antagonists have been deve-
surface receptors and intracellular signalling molecules. One of
loped as potential pharmaceuticals, for example, benzoxazinones,
(cyano)guanidines and (iso)quinoline carboxamides.8,9 A-740003, a
the cell surface receptors with upregulated expression in the
activated state of microglia is the purinergic P2X7 receptor.2,6 This
cyanoguanidine derivative, is a highly selective P2X7 receptor
receptor is thought to play a minimal role in normal physiology
antagonist that displays comparable IC50 values for both rat and
but has been shown to induce the release of inflammatory and
bioactive substances, which denote microglial activation.2,7
The P2X7 receptor comprises two transmembrane domains, an
aDepartment of Radiology and Nuclear Medicine, VU University Medical Center,
extracellular loop (with the adenosine triphosphate (ATP)
Amsterdam, The Netherlands
binding site) and N-terminal and C-terminal domains. Short
stimulation by binding of agonists (e.g. ATP) leads to formation bBV Cyclotron VU, Amsterdam, The Netherlands
of a channel permeable to small cations, whereas prolonged
*Correspondence to: Bieneke Janssen, Radionuclide Center, Department of
agonistic stimulation leads to a pore state that permits passage
Radiology and Nuclear Medicine, VU University Medical Center, De Boelelaan
of molecules up to 900 Da. Monif et al.2 proposed a model for
1085c, 1081 HV Amsterdam, The Netherlands.
the involvement of P2X7 receptors in the neuroinflammatory E-mail: b.janssen@vumc.nl
J. Label Compd. Radiopharm 2014, 57 509–516
Copyright © 2014 John Wiley & Sons, Ltd.

B. Janssen et al.
human P2X7 receptors (18 and 40 nM, respectively),10,11 which make it
a good candidate for translation from rodent to human. Therefore, A-
740003 is considered to be a promising compound for developing a
PET radiotracer for imaging of neuroinflammation. A-740003 is a
racemic compound and the affinity of the separate enantiomers
may differ.11 Nevertheless, for initial studies, racemic [11C]A-740003
was synthesised to enable preliminary in vivo studies on tracer
metabolism and biodistribution in healthy animals.
(1.08 g, 12.1 mmol) was added and the reaction was stirred at 60 °C
for 24 h. Another equivalent of both sodium dicyanamide and HCl
was added after 24 and 31 h and the reaction was stirred at 60 °C
for 16 more hours. The yellow precipitate was collected by filtration.
The precipitate was washed twice with cold water (10 mL) and twice
with cold Et2O (10 mL) and finally dried in a vacuum oven for 27 h at
40 °C to obtain N′′-cyano-N-5-quinolinyl guanidine (2) in 76% yield
(1.77 g, 7.17 mmol).
Rf value, 0.73 (DCM/methanol (MeOH), 9:1); 1H NMR (250.13 MHz,
DMSO-d6) δ 9.66 (s, 1H, NH), 9.09 (dd, 1H, J = 1.4 Hz, 4.5 Hz, CHAr), 8.72
(d, 1H, J = 8.5 Hz, CHAr), 8.01 (d, 1H, J = 8.4 Hz, CHAr), 7.75–7.83 (m, 3H,
CHAr), 7.37 (s, 2H, NH2); 13C NMR (125.78 MHz, DMSO-d6) δ 161.38
(Cguanidine), 149.26 (CHAr), 134.45 (CAr), 131.60 (CHAr), 124.78 (CHAr),
124.27 (CHAr), 122.00 (CHAr), 117.68 (CN); ESI-HRMS: calculated for
C11H9N5: 211.09; found 212.09 [M + H]+.
Experimental
General
Chemicals were obtained from commercial sources and used without further
purification. Solvents were purchased from Sigma-Aldrich (Zwijndrecht, the
Netherlands) and Biosolve (Valkenswaard, the Netherlands) and used as
received unless stated otherwise. Dichloromethane (DCM) and N,N-
dimethylformamide (DMF) were dried over 3 Å molecular sieves.
Tetrahydrofuran (THF) was first distilled from LiAlH4 and then stored on 3 Å
molecular sieves. Reactions were performed at room temperature unless
stated otherwise. Reactions were monitored by thin layer chromatography
on pre-coated silica 60 F254 aluminium plates (Merck, Darmstadt, Germany).
Spots were visualised by UV light, KMnO4, anisaldehyde, bromocresol green
or 4,4′-tetramethyldiamino-diphenylmethane-Cl2. Evaporation of solvents
was performed under reduced pressure at 40 °C using a rotary evaporator.
Flash column chromatography was performed manually on Silica gel 60 Å
(Merck, Darmstadt, Germany) or on a Büchi (Flawil, Switzerland) Sepacore
system (comprising a C-620 control unit, a C-660 fraction collector, two C-
601 gradient pumps and a C-640 UV detector) equipped with Büchi
Sepacore pre-packed flash columns. NMR spectroscopy was performed on
a Bruker (Billerica, MA, USA) Avance 250 (250.13 MHz for 1H and 62.90MHz
for 13C) or a Bruker Avance 500 (500.23 MHz for 1H and 125.78 MHz for 13C)
with chemical shifts (δ) reported in parts per million (ppm) relative to the
solvent (CDCl3, 1H 7.26ppm, 13C 77.16 ppm; CD3OD, 1H 3.31ppm, 13C
49.00 ppm; dimethyl sulfoxide (DMSO)-d6, 1H 2.50ppm, 13C 39.52 ppm).
Electrospray ionisation-high resolution mass spectrometry (ESI-HRMS) was
carried out using a Bruker microTOF-Q instrument in positive ion mode
(capillary potential of 4500 V). Analytical isocratic HPLC was performed on a
Jasco (Easton, MD, USA) PU-1580 station with a Xbridge C18 5 μm
(4.6 × 100 mm) column (Waters, Milford, MA, USA) using acetonitrile
(MeCN)/H2O (30:70, v/v) as eluent at a flow rate of 1 mL · minꢀ1 (method A),
a Jasco UV-2075 Plus UV detector (254 nm) and a NaI radioactivity detector
(Raytest, Straubenhardt, Germany). Chromatograms were acquired with
Raytest GINA Star software (version 5.01). Semi-preparative isocratic HPLC
was performed on a Jasco PU-1587 station with a Reprospher C18-DE 5 μm
(50 × 8 mm) column (Dr. Maisch, Ammerbuch-Entringen, Germany) using
MeCN/H2O/diisopropylamine (DIPA), (22:78:0.1, v/v/v) as eluent at a flow rate
of 3 mL · minꢀ1 (method B), a Jasco UV-1575 UV detector (254 nm), a custom-
made radioactivity detector and Jasco ChromNAV CFR software (version
1.14.01). Analytical HPLC for metabolite analysis was performed with Dionex
(Sunnyvale, CA, USA) UltiMate 3000 HPLC equipment with Chromeleon
software (version 6.8) on a Gemini C18 5-μm (10 × 250 mm) column
(Phenomenex, Torrance, CA, USA) with gradient and a mixture of MeCN (A)
and 0.1% trifluoroacetic acid in water (B) as eluent according to the following
scheme (method C): 0 min, 90% B at 0.25 mL · minꢀ1; 0.5 min, 90% Bꢀa1t
2-(3-(benzyloxy)-4-methoxyphenyl)acetic acid (4)
Benzyl bromide (150 μL, 1.26 mmol) was added to a solution of 2-(3-
hydroxy-4-methoxyphenyl)acetic acid (3; 203 mg, 1.11 mmol) and
tetrabutylphosphonium hydroxide (40% wt, 1.5 mL) in THF (2 mL) at 0 °C.
The reaction mixture was stirred for 3 h whilst warming to room
temperature. The reaction mixture was concentrated in vacuo and the
residue was dissolved in water (10 mL) and extracted with DCM
(3 × 10 mL). The aqueous layer was acidified to pH 1 with concentrated
HCl and extracted with DCM (3 × 10 mL). Combined organic layers were
dried over Na2SO4 and concentrated in vacuo. Flash column chroma-
tography was performed (hexanes/ethyl acetate (EtOAc) 2:1 + 0.1% acetic
acid) to obtain 2-(3-(benzyloxy)-4-methoxyphenyl)acetic acid (4) in 73%
yield (221 mg, 0.81 mmol).
Rf value, 0.36 (2% MeOH in DCM); 1H NMR (250.13 MHz, CDCl3) δ
7.28–7.45 (m, 5H, CHPh), 6.83–6.87 (m, 3H, CHAr), 5.13 (s, 2H, OCH2Ph),
3.87 (s, 3H, CH3), 3.54 (s, 2H, CH2COOH); 13C NMR (125.78 MHz, CDCl3) δ
177.03 (COOH), 149.23 (COCH2Ph), 148.35 (COCH3), 137.11 (CAr–benzyl),
128.67 (CHAr–Ph), 128.01 (CHAr–Ph), 127.58 (CHAr–Ph), 125.79 (CCH2COOH),
122.33 (CHAr), 115.44 (CHAr), 112.05 (CHAr), 71.23 (OCH2Ph), 56.19 (CH3),
40.51 (CH2COOH); ESI-HRMS: calculated for C16H16O4: 272.10; found
295.09 [M + Na]+.
2-(3-(benzyloxy)-4-methoxyphenyl)acetamide (5)
2-(3-(benzyloxy)-4-methoxyphenyl)acetic acid (4; 4.5 g, 16.5 mmol) was
dissolved in dry DCM (30 mL). Thionyl chloride (4 mL, 55 mmol) and a
drop of DMF were added and the mixture was stirred for 2 h, after which
the reaction mixture was concentrated in vacuo. The residue was
dissolved in dry THF (60 mL), and anhydrous ammonia gas was bubbled
through the solution for 5 min. A white precipitate was formed. The
reaction mixture was then partitioned in EtOAc (50 mL) and H2O
(30 mL). The aqueous layer was separated and extracted with EtOAc
(2 × 30 mL). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo to obtain 2-(3-(benzyloxy)-4-methoxyphenyl)
acetamide (5) in 92% yield (4.12 g, 15.2 mmol).
Rf value, 0.24 (2% MeOH in DCM); 1H NMR (250.13MHz, CDCl3) δ 7.27–7.44
(m, 5H, CHPh), 6.77–6.88 (m, 3H, CHAr), 5.57 (s, 1H, NH2), 5.32 (s, 1H, NH2), 5.14
(s, 2H, OCH2Ph), 3.88 (s, 3H, CH3), 3.46 (s, 2H, CH2C(O)NH2); 13C NMR
(125.78 MHz, CDCl3) δ 173.99 (C¼O), 149.27 (COCH2Ph), 148.43 (COCH3),
136.93 (CAr–benzyl), 128.69 (CHAr–Ph), 128.06 (CHAr–Ph), 127.54 (CHAr–Ph), 127.31
(CCH2C(O)NH2), 122.36 (CHAr), 115.47 (CHAr), 112.34 (CHAr), 71.13 (OCH2Ph),
56.12 (CH3), 42.91 (CH2C(O)NH2); ESI-HRMS: calculated for C16H17NO3:
271.12; found 272.13 [M + H]+, 294.11 [M+Na]+.
4mL· minꢀ1; 9.0min, 30% B at 4mL· minꢀ1; 13.0 min, 30% B at 4 mL · min
14.0 min, 90% B at 4 mL · minꢀ1; and 15.0 min, 90% B at 0.25 mL · minꢀ1
;
.
Healthy male Wistar rats were obtained from Harlan Netherlands B.V.
(Horst, the Netherlands). All animal experiments were performed according
to the National Institute of Health principles of laboratory animal care and N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-(benzyloxy)-4-
Dutch national law (‘Wet op de proefdieren’, Stb 1985, 336).
methoxyphenyl)acetamide (6)
To a suspension of 2-(3-(benzyloxy)-4-methoxyphenyl)acetamide (5; 4.12g,
15.2 mmol), 1H-benzotriazole (1.81g, 15.2mmol) and pivalaldehyde
(3.5mL, 32mmol) in toluene (50mL) on MgSO4 (3.0g, 25 mmol),
p-toluenesulphonic acid (144 mg, 0.8 mmol) was added. After refluxing
Synthesis
N′′-cyano-N-5-quinolinyl guanidine (2)11
Water (6 mL) was added to 5-quinolinamine (1; 1.35 g, 9.37 mmol) and overnight, the reaction mixture was cooled at room temperature and
6 M HCl (2 mL) to give a dark red solution. Sodium dicyanamide DCM (30 mL) was added. MgSO4 was filtered off and the filtrate was
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J. Label Compd. Radiopharm 2014, 57 509–516

B. Janssen et al.
concentrated in vacuo. Flash column chromatography was performed
(20–50%, EtOAc/hexanes+ 0.1% triethylamine) to obtain N-(1-(1H-
benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-(benzyloxy)-4-methoxyphenyl)
acetamide (6) in 74% yield (5.12 g, 11.2mmol).
N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-((tert-butyldi-
methylsilyl)oxy)-4-methoxyphenyl)acetamide (10)
N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-2,2-dimethylpropyl)-2-(3-hydroxy-4-
methoxyphenyl)-acetamide (9; 1.8 g, 4.9 mmol) and tert-butyldime-
thylchlorosilane (TBDMSCl) (1.5 g, 9.9 mmol) were dissolved in dry DCM
(70 mL). Imidazole (1.0 g, 14 mmol) was added and the mixture was stirred
overnight. After addition of water (70 mL), the mixture was partitioned
between water and DCM. The water layer was extracted with DCM
(50 mL). The combined organic layers were washed with brine (100 mL), dried
over Na2SO4 and concentrated in vacuo. Flash column chromatography
(0.5% MeOH in DCM) was performed to obtain N-(1-(1H-benzotriazol-1-yl)-
2,2-dimethylpropyl)-2-(3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)
acetamide (10) in 87% yield (2.0 g, 4.2 mmol).
Rf value, 0.53 (EtOAc/hexanes, 1:1); 1H NMR (250.13 MHz, CDCl3) δ 8.04
(d, 1H, J= 8.3Hz, CHAr), 7.65 (d, 1H, J = 8.3 Hz, CHAr), 7.27–7.51 (m, 7H, CHPh
+
CHAr), 6.76–6.90 (m, 4H, CHAr + NH), 6.44 (d, 1H, J = 9.8Hz, CH), 5.09 (q, 2H,
J= 3.0 Hz, 12.2 Hz, OCH2Ph), 3.89 (s, 3H, CH3), 3.49 (q, 2H, J= 8.7Hz,
16.2 Hz, CH2C(O)NH2), 0.93 (s, 9H, 3x CH3); 13C NMR (125.78 MHz, CDCl3) δ
171.02 (C¼O), 149.24 (CAr), 148.68 (CAr), 144.86 (CAr), 136.82 (CAr), 133.75
(CAr), 128.58 (CHAr), 127.92 (CHAr), 127.71 (CHAr), 127.33 (CHAr), 126.25
(CAr), 124.06 (CHAr), 122.21 (CHAr), 119.75 (CHAr), 114.85 (CHAr), 112.26 (CHAr),
109.95 (CHAr), 71.04 (OCH2Ph), 68.80 (CHC(CH3)3), 56.06 (CH3), 42.96 (CH2),
37.04 (C(CH3)3), 25.93 (C(CH3)3); ESI-HRMS: calculated for C27H30N4O3:
458.23; found 459.24 [M + H]+, 481.22 [M + Na]+.
Rf value, 0.40 (1% MeOH in DCM); 1H NMR (500.23 MHz, CDCl3) δ 8.06
(d, 1H, J = 8.2 Hz, CHAr), 7.68 (d, 1H, J = 8.2 Hz, CHAr), 7.49–7.53 (m, 1H,
CHAr), 7.36–7.39 (m, 1H, CHAr), 6.89 (d, 1H, J = 8.2 Hz, NH), 6.77–6.81 (m,
3H, CHAr), 6.46 (d, 1H, J = 9.8 Hz, CH), 3.84 (s, 3H, CH3), 3.53 (q, 2H,
J = 16.4 Hz, 54.2 Hz, CH2), 1.01 (s, 9H, Si(CH3)2C(CH3)3), 0.96 (s, 9H, C(CH3)
3), 0.17 (d, 6H, J = 4.1 Hz, Si(CH3)2C(CH3)3); 13C NMR (125.78 MHz, CDCl3)
δ 171.04 (C¼O), 150.61 (CAr), 145.59 (CAr), 144.86 (CAr), 133.72 (CAr),
127.66 (CHAr), 126.30 (CAr), 124.00 (CHAr), 122.68 (CHAr), 122.00 (CHAr),
119.75 (CHAr), 112.55 (CHAr), 109.94 (CHAr), 68.66 (CH(C(CH3)3), 55.48
(CH3), 42.82 (CH2), 37.07 (C(CH3)3), 25.91 (Si(CH3)2C(CH3)3), 25.69
(C(CH3)3), 18.45 (Si(CH3)2C(CH3)3), ꢀ4.64 (Si(CH3)2C(CH3)3); ESI-HRMS:
calculated for C26H38N4O3Si: 482.27; found 483.28 [M + H]+.
2-(3-(benzyloxy)-4-methoxyphenyl)-N-(1-(2-cyano-3-(quinolin-5-yl)
guanidino)-2,2-dimethylpropyl)acetamide (7)
N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-(benzyloxy)-4-
methoxyphenyl)acetamide (6; 593 mg, 1.29 mmol) and N′′-cyano-N-5-
quinolinyl guanidine (2; 282 mg, 1.34 mmol) were dissolved in dry DMF
(10 mL) and treated with finely powdered Cs2CO3, (1.09 g, 3.34 mmol).
After stirring for 25 h, the brown solution was partitioned between EtOAc
(20 mL) and H2O (10 mL), and the aqueous layer was extracted twice with
EtOAc (20 mL). The combined organic layers were washed with water
(10 mL) once, dried over Na2SO4 and concentrated in vacuo. Flash column
chromatography was performed twice (1–5%, MeOH/DCM) to obtain 2-(3-
(benzyloxy)-4-methoxyphenyl)-N-(1-(2-cyano-3-(quinolin-5-yl)
N-(1-(2-cyano-3-(quinolin-5-yl)guanidino)-2,2-dimethylpropyl)-2-(3-
hydroxy-4-methoxyphenyl)acetamide (8)
N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-((tert-butyldimethylsilyl)
oxy)-4-methoxyphenyl)acetamide (10; 2.0 g, 4.1mmol) and N′′-cyano-N-5-
quinolinyl guanidine (2; 0.9 g, 4.3 mmol) were dissolved in dry DMF
(30 mL) and treated with finely powdered Cs2CO3 (3.5g, 10.7mmol). After
stirring for 25 h, the solution was partitioned between EtOAc (50 mL) and
H2O (10 mL) and the aqueous layer was extracted twice with EtOAc
(50mL). The combined organic layers were washed with brine (2× 20mL),
dried over Na2SO4 and concentrated in vacuo. Flash column chromato-
graphy was performed (1–5%, MeOH/DCM) to obtain N-(1-(2-cyano-3-
(quinolin-5-yl)guanidino)-2,2-dimethylpropyl)-2-(3-hydroxy-4-methoxyphenyl)
acetamide (8) as an off-white solid in 29% yield (0.56 g, 1.2 mmol).
Rf value, 0.21 (5% MeOH in DCM); 1H NMR (500.23 MHz, DMSO-d6) δ
9.61 (s, 1H, NH), 8.94 (d, 1H, J = 2.8 Hz, CHAr), 8.89 (s, 1H, OH), 8.27
(d, 1H, J = 8.5 Hz, CHAr), 8.02 (s, 1H, NH), 7.97 (d, 1H, J = 8.2 Hz, CHAr),
7.75 (t, 1H, J = 8.2 Hz, CHAr), 7.55–7.57 (m, 1H, CHAr), 7.42 (d, 1H,
J = 7.3 Hz, CHAr), 6.83 (d, 1H, J = 8.2 Hz, CHAr), 6.63–6.72 (m, 3H, CHAr + NH),
5.52 (t, 1H, J = 9.1 Hz, CH), 3.73 (s, 3H, CH3), 3.39 (q, 2H, J = 13.8 Hz, CH2),
0.89 (s, 9H, C(CH3)3); 13C NMR (125.78 MHz, DMSO-d6) δ 170.97 (C¼O),
158.71 (Cguanidine), 150.74 (CHAr), 148.25 (CAr), 146.35 (CAr), 146.23 (CAr),
133.15 (CAr), 131.21 (CHAr), 129.13 (CHAr), 128.42 (CAr), 127.85 (CHAr),
124.69 (CAr), 124.32 (CHAr), 121.53 (CHAr), 119.56 (CHAr), 116.44 (CHAr),
116.43 (CN), 112.09 (CHAr), 64.56 (CH(C(CH3)3), 55.64 (CH3), 41.52
(CH2), 35.80 (C(CH3)3), 25.26 (C(CH3)3); ESI-HRMS: calculated for
guanidino)-2,2-dimethylpropyl)acetamide (7) as a light orange solid in
47% yield (0.34 g, 0.61 mmol).
Rf value, 0.26 (2% MeOH in DCM); H NMR (500.23 MHz, CDCl3) δ 9.74
1
(s, 1H, NH), 8.94 (dd, 1H, J = 1.3 Hz, 4.1 Hz, CHAr), 8.24 (d, 1H, J = 8.5 Hz,
CHAr), 8.06 (d, 1H, J = 8.5 Hz, CHAr), 7.70 (t, 1H, J = 7.9 Hz, CHAr), 7.53
(d, 1H, J = 7.6 Hz, CHAr), 7.41–7.44 (m, 3H, CHAr), 7.33–7.38 (m, 3H, CHAr),
6.83–6.92 (m, 3H, CHAr), 6.55 (s, 1H, NH), 5.89 (s, 1H, NH), 5.19 (t, 1H,
J = 9.1 Hz, CH), 5.14 (s, 2H, OCH2Ph), 3.92 (s, 3H, CH3), 3.59 (s, 2H, CH2),
0.96 (s, 9H, 3x CH3); 13C NMR (125.78 MHz, CDCl3) δ 160.08 (Cguanidine),
150.60 (CHAr), 149.48 (CAr), 148.66 (CAr), 148.62 (CAr), 136.58 (CAr), 130.86
(CHAr), 128.85 (CHAr), 128.62 (CHAr), 128.09 (CHAr), 127.21 (CHAr), 125.62
(CAr), 124.27 (CAr) 122.18 (CHAr), 121.37 (CHAr), 117.22 (CN), 115.01 (CHAr),
112.29 (CHAr), 70.99 (OCH2Ph), 65.75 (CH(C(CH3)3)), 56.06 (CH3), 42.70
(CH2), 34.94 (C(CH3)3)), 25.48 (C(CH3)3); ESI-HRMS: calculated for
C32H34N6O3: 550.27; found 551.28 [M + H]+.
N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-hydroxy-4-
methoxyphenyl)acetamide (9)
N-(1-(1H-benzotriazol-1-yl)-2,2-dimethylpropyl)-2-(3-(benzyloxy)-4-
methoxyphenyl)acetamide (6; 2.4 g, 5.2 mmol) was dissolved in DCM
(20 mL). Pd/C (134 mg, 1.3 mmol) was added, and the flask was
sealed with a septum and left stirring overnight under hydrogen
atmosphere. The reaction mixture was passed over Celite and
concentrated in vacuo. Flash column chromatography (2–6%
MeOH/DCM) was performed to obtain N-(1-(1H-benzotriazol-1-yl)-
2,2-dimethylpropyl)-2-(3-hydroxy-4-methoxyphenyl)acetamide (9) in
94% yield (1.8 g, 4.9 mmol).
C
25H28N6O3: 460.22; found 461.21 [M + H]+.
A-740003
Reference compound N-(1-{[(cyanoimino)(5-quinolinylamino)methyl]
amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide (A-740003)
Rf value, 0.47 (DCM/MeOH, 9:1); 1H NMR (500.23 MHz, CDCl3) δ 8.06 was synthesised with an overall yield of 7% (0.62 g, 1.3mmol) as described
(d, 1H, J = 8.5 Hz, CHAr), 7.68 (d, 1H, J = 8.5 Hz, CHAr), 7.35–7.39 (m, 1H, CHAr) by Perez-Medrano et al.,11 except for some minor changes: compound 2
7.49–7.52 (m, 1H, CHAr), 6.95 (d, 1H, J = 9.8 Hz, NH), 6.82–6.86 (m, 2H, CHAr), was synthesised as described above, ammonia gas was used in the
6.72 (dd, 1H, J = 1.9 Hz, 7.9 Hz, CHAr), 6.49 (d, 1H, J = 9.8 Hz, CH), 5.94 (s, 1H,
conversion from carboxylic acid to amide and DMF was used as solvent in
OH), 3.90 (s, 3H, CH3), 3.54 (q, 2H, J = 16.4 Hz, 27.5 Hz, CH2), 0.99 (s, 9H, 3x the coupling of compound 2 to N-(1-(1H-benzo[d][1,2,3]triazol-1-yl)-2,2-
CH3); 13C NMR (125.78 MHz, CDCl3) δ 171.12 (C = O), 146.20 (CAr), 146.17
(CAr), 144.78 (CAr), 133.75 (CAr), 127.74 (CHAr), 126.90 (CAr), 124.10 (CHAr),
120.99 (CHAr), 119.69 (CHAr), 115.60 (CHAr), 111.22 (CHAr), 110.00
(CHAr), 68.84 (CH(C(CH3)3)), 55.98 (CH3), 42.82 (CH2), 37.10 (C(CH3)3),
25.94 (C(CH3)3); ESI-HRMS: calculated for C20H24N4O3: 368.18; found
369.19 [M + H]+.
dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide to yield A-740003.
Rf value, 0.48 (DCM/MeOH, 9:1); 1H NMR (500.23 MHz, CDCl3) δ 9.71 (s,
1H, NH), 8.93 (d, 1H, J = 4.1 Hz, CHAr), 8.22 (d, 1H, J = 8.5 Hz, CHAr), 8.05 (d,
1H, J = 8.5 Hz, CHAr), 7.69 (t, 1H, J = 8.5 Hz, CHAr), 7.50 (d, 1H, J = 7.6 Hz,
CHAr), 7.39–7.42 (m, 1H, CHAr), 6.79–6.87 (m, 4H, CHAr + NH), 5.97 (s, 1H,
NH), 5.24 (t, 1H, J = 9.2 Hz, CH), 3.89 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.52
J. Label Compd. Radiopharm 2014, 57 509–516
Copyright © 2014 John Wiley & Sons, Ltd.
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B. Janssen et al.
(s, 2H, CH2), 1.01 (s, 9H, C(CH3)3); 13C NMR (125.78 MHz, CDCl3) δ 160.15
(Cguanidine), 150.66 (CHAr), 149.51 (CAr), 148.70 (CAr), 130.89 (CHAr),
Results and discussion
128.86 (CHAr), 125.92 (CAr), 121.61 (CHAr), 121.41 (CHAr), 117.33 (CN), Synthesis
112.29 (CHAr), 111.66 (CHAr), 68.74 (CH(C(CH3)3)), 55.96 (CH3), 42.78
Reference compound A-740003 was synthesised according to a
procedure previously described by Perez-Medrano et al.11 The
synthesis route towards precursor 8, depicted in Scheme 1, was
adapted from this procedure. In order to be able to incorporate
a [11C]methyl group at the phenolic hydroxyl of precursor 8,
use of a protective group at this position that would withstand
all reaction conditions throughout the synthesis and could be
removed at the last step was required. Allyl and benzyl
protecting groups were selected for this purpose.12 The latter
was favoured on the basis of test reactions in which
deprotection of benzyl-protected acetamide 5 or allyl protected
acetamide (structure not shown) was performed. The synthesis
of benzyl-protected precursor 7 was started with reaction of 5-
aminoquinoline 1 with sodium dicyanamide in water in the
presence of HCl resulting in N′′-cyano-N-5-quinolinyl guanidine
2 in 76% yield. The phenolic hydroxyl group of 2-(3-hydroxy-4-
methoxyphenyl)acetic acid 3 was protected with benzyl bromide
in the presence of tetrabutylphosphonium hydroxide resulting in
carboxylic acid 4 in 73% yield.13 Carboxylic acid 4 was then
converted to acetamide 5 in 92% yield by reaction with thionyl
chloride followed by treatment of the acid chloride intermediate
with ammonia. Reaction of acetamide 5 with pivalaldehyde and
benzotriazole yielded compound 6 in 74%. Subsequently,
compound 6 was coupled to N′′-cyano-N-5-quinolinyl guanidine
2 in the presence of Cs2CO3 to obtain the benzyl-protected
precursor 7 in a yield of 47%. Unfortunately, despite the use of
several deprotection methods, precursor 8 could not be
obtained. The following deprotection conditions were tested:
Pd/C with hydrogen, trifluoroacetic acid, titanium trichloride
and magnesium, boron trifluoride etherate and sodium iodide
and aluminium chloride.14–16 In all conditions tested, the
formation of many different unidentified products as well as
reduction of benzyl protected precursor 7 at undesired positions
was observed by mass spectrometry. Therefore, an alternative
synthesis route towards precursor 8 was developed, starting with
benzyl deprotection of 6 with hydrogen and Pd/C. Subsequently,
the phenolic hydroxyl group of compound 9 was protected with
TBDMSCl, in the presence of imidazole in a yield of 77%. TBDMS
was selected for its mild and selective cleavage. TBDMS-protected
compound 10 was then coupled to N′′-cyano-N-5-quinolinyl
guanidine 2, during which unforeseen but complete TBDMS
deprotection occurred, resulting in precursor 8 in 29% yield.
TBDMS deprotection was probably because of the presence of
trace amounts of water.17,18 All other yields obtained were in
(CH2), 35.07 (C(CH3)3), 25.56 (C(CH3)3); ESI-HRMS: calculated for
C26H30N6O3: 474.24; found 475.24 [M + H]+.
Radiosynthesis
[11C]A-740003
[
11C]CO2 was produced by the 14N(p,α)11C nuclear reaction performed in
a 0.5% O2/N2 gas mixture using an IBA Cyclone 18/9 cyclotron (IBA,
Louvain-la-Neuve, Belgium). [11C]CO2 was trapped in 100 μL of 0.1 M
LiAlH4 in THF. After evaporation of THF by heating the reaction vial to
130 °C under a helium flow, 250 μL 60% HI in water was added. Formed
[
11C]CH3I was then distilled to the second reaction vial containing a
solution of precursor 8 (0.5 mg, 1.1 μmol) and tetrabutylammonium
hydroxide, (1 μL, 1.5 μmol, 40% solution in water) in 250 μL of DMSO.
The reaction mixture was heated at 70 °C for 5 min, after which it was
diluted with 0.8 mL of water and purified by semi-preparative HPLC
(method B). The fraction containing [11C]A-740003 (tR = 11.4 min) was
collected, diluted with 40 mL of water and trapped on a Sep-Pak Plus
tC18 cartridge (Waters, Milford, MA, USA), preconditioned with 10 mL of
ethanol and 10 mL of water. The cartridge was washed with 20 mL of
water and subsequently [11C]A-740003 was eluted with 1 mL of EtOH
and diluted with 5 mL of 7.09 mM NaH2PO4 in saline. [11C]A-740003 was
obtained in a decay-corrected radiochemical yield of 26–59% (n = 2) from
end of bombardment with a radiochemical purity >97%, a specific
activity (SA) of 41 22 GBq/μmol (n = 3) and an overall synthesis time of
approximately 50 min. The identity of the product was confirmed
with analytical HPLC by coinjection of the product and non-labelled
A-740003 (method A, tR = 7.4 min).
Biodistribution
Healthy male Wistar rats (210–275 g) were injected with approximately
50 MBq (at start of experiment) [11C]A-740003 in the tail vein under
isoflurane anaesthesia. Rats were sacrificed and dissected at 5, 15, 30
and 60 min post-injection (n = 4 for 5 and 30 min and n = 3 for 15 and
60 min). Blood, heart, lungs, liver, kidneys, olfactory bulbs, hippocampus,
striatum, cerebral cortex, cerebellum and the rest of the brain were
collected, weighed and counted for radioactivity in a Wallac Universal
Gamma Counter 1282 (PerkinElmer, Waltham, MA, USA). Biodistribution
data were expressed as percentage injected dose per gram tissue (%ID/g).
Metabolite analysis
Healthy male Wistar rats (230–250 g) were injected with approximately
100 MBq (at start of experiment) [11C]A-740003 in the tail vein under
isoflurane anaesthesia. Rats were sacrificed at 15 and 45 min (n = 3 per
time point). Blood samples were obtained, and the brain was removed. accordance with those reported in literature.
Blood samples were collected in heparin tubes (BD Vacutainer NH 119
I.U., 7 mL, BD Vacitainer Systems, Plymouth, UK) and centrifuged at
Radiosynthesis
4000 rpm for 5 min at 4 °C (Hettich Universal 32, Andreas Hettich GmbH
& Co. KG, Tuttlingen, Germany). The brain was put in a falcon tube In order to obtain [11C]A-740003, the phenolic hydroxyl group of
containing saline (5 mL) and homogenised with a disperser (IKA T18 B
Ultra-Turrax, IKA®-Werke GmbH & Co. KG, Staufen, Germany) before
centrifugation (5 min, 4000 rpm, 20 °C, Hettich Universal 32). Plasma
and brain supernatant were separated from blood cells and brain
precipitate, respectively. Plasma and brain supernatant were loaded onto
a Sep-Pak tC18 cartridge, followed by washing with 10 mL of water to
obtain the polar fraction. The non-polar fraction was then eluted with
1.5 mL of MeOH and 1.5 mL of water. All separate fractions were counted
for radioactivity in a Wizard Gamma Counter 1470 or 2480 (Wallac/
PerkinElmer, Waltham, MA, USA) and the non-polar fraction was further
analysed by HPLC using method C.
precursor 8 was subjected to carbon-11 methylation by reaction
with [11C]methyl iodide in the presence of a base (Scheme 2).
Although radiolabelling of phenolic hydroxyl groups with [11C]
methyl iodide is often performed in the presence of sodium
hydroxide,19–23 this was not successful for precursor 8 using
either DMF or DMSO as a solvent (Table 1). However, using the
milder base tetrabutylammonium hydroxide, conversion of
[11C]methyl iodide to [11C]A-740003 was on average 87% (n = 4,
determined as percentage of the total activity of the crude
reaction mixture observed on analytical HPLC). During
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J. Label Compd. Radiopharm 2014, 57 509–516

B. Janssen et al.
Scheme 1. Synthesis route towards precursor 8. Reagents and conditions: a) NaN(CN)2, HCl, H2O, 60 °C, 47 h, 76%; b) n-Bu4POH, BnBr, THF, 3 h, 73%; c) SOCl2, DMF, DCM,
1 h; d) NH3, THF, 5 min, 92% (over two steps); e) 1H-benzotriazole, pivalaldehyde, pTsOH, MgSO4, toluene, reflux, 16 h, 74%; f) 2, Cs2CO3, DMF, 25 h, 47%; g) several benzyl
deprotection methods; h) H2, Pd/C, DCM, 16 h, 94%; i) TBDMSCl, imidazole, DCM, 16 h, 87%; j) 2, Cs2CO3, DMF, 25 h, 29%.
Scheme 2. Radiosynthesis of [11C]A-740003.
purification of the radiolabelled product by means of semi- was formulated in 6 mL of 17% EtOH in 7.09 mM NaH2PO4 in
preparative HPLC, some difficulties (e.g. no retention of saline to obtain [11C]A-740003 (2.9–5.4 GBq) with sufficient
radiolabelled product) were encountered that could be solved radiochemical purity (>97%) and SA (41 22 GBq/μmol) to be
by dilution of the reaction mixture with 0.8 mL water prior to evaluated in vivo. Formulated [11C]A-740003 was determined to
semi-preparative HPLC purification. Subsequently, the product be stable until injection by analytical HPLC.
J. Label Compd. Radiopharm 2014, 57 509–516
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

B. Janssen et al.
Table 1. Optimisation radiolabelling [11C]A-740003
Conversion on
analytical HPLC
Solvent (250 μL)
Base
T (°C)
RCY (d.c.)
SA (GBq/μmol)
DMF
DMSO
DMSO
NaOH (5 μmol)
NaOH (5 μmol)
TBAOH (1.5 μmol)
80
70
70
<1% (n = 1)
<1% (n = 2)
n.d.
n.d.
n.d.
n.d.
87 6% (n = 4)a
26–59% (n = 2)b
41 22 (n = 3)b
Precursor concentration = 4 mM; reaction time = 5 min.
n.d., not determined.
aConversion was not determined in tracer productions for animal experiments.
bBecause of optimisation of purification, only two syntheses were identically performed technically.
Metabolite analysis
although excretion via intestines could still be possible. At all
time points, tracer uptake observed in brain regions was low
and even close to background measurements.
To assess in vivo stability of [11C]A-740003, healthy male Wistar
rats were injected with around 100 MBq of the formulated
radiolabelled compound. The amount of intact [11C]A-740003
in plasma was determined to be 59% and 31% at 15 and
45 min after injection, respectively (Table 2). Two unidentified
non-polar metabolites were observed on HPLC as can be seen
in Figure 1.
In brain supernatant, the measured amount of non-polar
radioactive compound was 8% and 3% at 15 and 45 min
after injection, respectively. The percentage of intact tracer
in the non-polar fraction could not be identified with HPLC,
because the amount of activity in the brain was too low.
Furthermore, the amount of polar metabolites was sub-
stantially higher in the brain than in plasma, which is highly
unlikely. This might be explained by the low brain uptake of
the parent tracer (as seen in the biodistribution study,
Figure 2), causing a distorted view due to radioactive species
remaining in the blood vessels of the brain. Radioactivity in
the brain could therefore essentially be allocated to blood
born polar metabolites.
High variability in SA of [11C]A-740003 between experiments
was observed (5–81 GBq/μmol, corresponding with injection of
0.3–4.7 μg of carrier). With lower SA, more carrier was injected,
which may have affected pharmacokinetics. Overall, higher
values would be expected with lower SA due to longer
circulation in the blood. To correct for the variable SA, organ-
to-blood ratios were calculated as depicted for the brain in
Figure 3. These data suggest that there is a slight increase in
tracer uptake over time or that clearance of radioactivity is faster
in blood than in brain due to radioactive metabolites present
in blood.
The low brain uptake indicates that either the tracer does not
enter the brain through the blood–brain barrier or that it is not
retained in the brain due to the low expression level of P2X7
receptors in healthy subjects. P2X7 receptor expression,
however, was shown to be widespread in rat brain by means
of autoradiography, with moderate-high tritiated ligand binding
in several brain regions, including cerebral cortex, hippocampus
and olfactory nucleus, and a maximum number of receptor
binding sites (Bmax) in brain of 112 fmol · mgꢀ1 has been
reported.24 For comparison, Bmax values for the metabotropic
glutamate 5 (mGlu5) receptor in rat brain are twofold to
Biodistribution
eightfold higher and range from 231 to 870 fmol · mgꢀ1 25–27
In
.
In the biodistribution study, around 50 MBq of formulated [11C]
A-740003 was injected in healthy male Wistar rats. [11C]A-
740003 showed clearance via kidneys and liver (Figure 2),
recent years, successful radiopharmaceuticals for the mGlu5
receptor have been published.26,28 Uptake of these
radiopharmaceuticals in whole rat brain at 30 min post-injection
was 0.074, 0.057 and 0.128%ID/cc for [11C]M-PEPy, [11C]M-MPEP
and [11C]MPEP, respectively.28 For [11C]ABP688, uptake was
0.19 and 0.22%ID/g in hippocampus and striatum, respectively.26
The observed lower brain uptake of [11C]A-740003 (about 0.03%
ID/g at 30 min post-injection, Figure 2) might therefore be
explained by the lower Bmax value for the P2X7 receptor
compared with the mGlu5 receptor. Nevertheless, the brain
region-to-blood ratios suggest a slow but steady increase of
tracer over time.
Table 2. Metabolite analysis of [11C]A-740003 in plasma and
brain supernatant
Plasma
Brain
15 min 45 min
15 min
(%)
45 min
(%)
(%)
(%)
In summary, [11C]A-740003 was successfully synthesised and,
in healthy male Wistar rats, low brain uptake and a moderate
metabolic rate were observed. To the best of our knowledge,
[11C]A-740003 is the first potential PET tracer for the P2X7
receptor that has been synthesised and evaluated in vivo. Further
studies are needed to assess whether [11C]A-740003 shows
higher brain uptake in animal models of neuroinflammation with
increased P2X7 receptor expression, thus to evaluate its potential
as a novel PET tracer.
Non-polar fraction
Intact tracer
Non-polar
metabolites
Polar metabolites
70
59
11
6
7
1
43
31
12
2
2
1
8
1%
n.d.
n.d.
3
n.d.
n.d.
1%
30
6
57
2
92 1%
97 2%
n.d., not determined.
n = 3 for all time points.
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 509–516

B. Janssen et al.
Figure 1. Radiochromatogram of the non-polar fraction of plasma after 15 min. Intact [11C]A-740003 has a retention time of 10.1 min.
Acknowledgements
The research leading to these results has received funding from
the European Union’s Seventh Framework Programme (FP7/
2007-2013) under grant agreement no. HEALTH-F2-2011-278850
(INMiND). J. Verbeek is acknowledged for his assistance during
[11C]methylation experiments.
Conflict of interest
The authors did not report any conflict of interest.
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J. Label Compd. Radiopharm 2014, 57 509–516