Preclinical Evaluation of the First Adenosine A1 Receptor Partial Agonist Radioligand for Positron Emission Tomography Imaging

Article abstract of DOI:10.1021/acs.jmedchem.8b01009

Central adenosine A₁ receptor (A₁R) is implicated in pain, sleep, substance use disorders, and neurodegenerative diseases, and is an important target for pharmaceutical development. Radiotracers for A₁R positron emission t

Full text of DOI:10.1021/acs.jmedchem.8b01009

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Article
1
Preclinical evaluation of the first adenosine A receptor partial
agonist radioligand for positron emission tomography (PET) imaging
Min Guo, Zhan-Guo Gao, Ryan Tyler, Tyler Stodden, Yang Li, Joseph Ramsey,
Wen-Jing Zhao, Gene-Jack Wang, Corinde E. Wiers, Joanna S. Fowler,
Kenner C. Rice, Kenneth A. Jacobson, Sung Won Kim, and Nora D. Volkow
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01009 • Publication Date (Web): 25 Oct 2018
Downloaded from http://pubs.acs.org on October 25, 2018
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Journal of Medicinal Chemistry
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Preclinical evaluation of the first adenosine A1 receptor partial
agonist radioligand for positron emission tomography (PET) imaging
†
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‡
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Min Guo , Zhan-Guo Gao , Ryan Tyler , Tyler Stodden , Yang Li , Joseph Ramsey , Wen-Jing Zhao ,
†
†
†
#
‡
Gene-Jack Wang , Corinde E. Wiers , Joanna S. Fowler , Kenner C. Rice , Kenneth A. Jacobson , Sung
Won Kim†,*, Nora D. Volkow†,§,*
† Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda,
Maryland 20892-1013, United States
‡ Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, Maryland 20892-0810, United States
# Drug Design and Synthesis Section, National Institute on Drug Abuse and National Institute on Alcohol Abuse and
Alcoholism, National Institutes of Health, Rockville, Maryland 20892, United States
§ National Institute on Drug Abuse, National Institutes of Health, Bethesda, Maryland 20892-1013, United States
KEYWORDS: Adenosine A1 Receptor, Positron Emission Tomography, Blood-brain Permeability, Partial Agonist, Rodents
ABSTRACT: Central adenosine A1 receptor (A1R) is implicated in pain, sleep, substance use disorders,
and neurodegenerative diseases, and is an important target for pharmaceutical development. Radiotracers
for A1R positron emission tomography (PET) would enable measurement of dynamic interaction of
endogenous adenosine and A1R during the sleep-awake cycle. Although several human A1R PET tracers
have been developed, most are xanthine-based antagonists that failed to demonstrate competitive binding
against endogenous adenosine. Herein, we explored nonnucleoside (3,5-dicyanopyridine and 5-
cyanopyrimidine) templates for developing an agonist A1R PET radiotracer. We synthesized novel
analogues, including 2-amino-4-(3-methoxyphenyl)-6-(2-(6-methylpyridin-2-yl)ethyl)pyridine-3,5-
dicarbonitrile (MMPD, 22b), a partial A1R agonist of sub-nanomolar affinity. [11C]22b showed suitable
blood-brain barrier (BBB) permeability and test-retest reproducibility. Regional brain uptake of [11C]22b
was consistent with known brain A1R distribution and was blocked significantly by A1R but not A2AR
ligands. [11C]22b is the first BBB permeable A1R partial agonist PET radiotracer with the promise of
detecting endogenous adenosine fluctuations.
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INTRODUCTION
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Adenosine is a nucleoside neuromodulator that modulates release of other major neurotransmitters in
brain such as dopamine, acetylcholine, glutamic acid, etc.1-3 The neuroprotective, anxiolytic, and
antiepileptic effects of adenosine appear to be mediated mainly through the adenosine A1 receptor (A1R).
The A1R is expressed in neurons and glial cells and is highly abundant throughout the brain including the
cerebral cortex, hippocampus and striatum.4 A1R activation attenuates neurotransmitter release and
inhibits adenylyl cyclase activity reducing cAMP production and lowering synaptic transmission. Since
A1R has been considered a potential drug target for Alzheimer’s disease, cerebral ischemia, epilepsy and
substance use disorders, functionally selective and highly potent ligands have been extensively explored
over the last two decades.5-6
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Positron emission tomography (PET) radiotracers targeting A1R have been developed as quantitative
imaging tools to sensitively and non-invasively delineate pathological changes in vivo in humans. Among
A1R radioligands,7
a
xanthine derivative, [1-methyl-11C]8-dicyclopropylmethyl-1-methyl-3-
propylxanthine ([11C]MPDX)8 was the first C-11 based radioligand studied in healthy human subjects9-10
and subsequently in patients with Alzheimer’s disease (AD),11 showing region-specific decreases in A1R
with normal aging and AD. An F-18 labeled xanthine analogue, [18F]CPFPX (3, Figure 1A) was also
developed12-13 and tested in healthy controls13 and revealed an elevation of its binding to A1R with sleep
deprivation in humans.14-15 A nonxanthine-based radiotracer, 2-[1-(methyl-11C)-4-piperidinyl]-6-(2-
phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone ([11C]FR194921) showed acceptable blood-
brain barrier (BBB) permeability but relatively low specific binding in the rodent brain.16
In this study we were particularly interested in a tracer that would allow us to measure changes in
extracellular adenosine levels in the brain and A1R signaling. This required a radioligand with good BBB
penetration, specificity for A1R and binding that was sensitive to changes in endogenous adenosine.
Previously, displacement studies of [11C]MPDX binding either by increasing adenosine levels17 or by
agonist pretreatment18 were not successful presumably due to its inverse agonist binding characteristics.
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Although an agonist ligand for A1R would be expected to have a higher sensitivity to changes in
endogenous adenosine levels than an antagonist,19-20 to date, no BBB permeable A1R agonist PET
radioligand has been reported. The majority of synthetic antagonists have been designed based on a natural
xanthine antagonist, caffeine, or a on a pyrazolotriazolopyrimidine scaffold (Figure 1A).5-6 The
endogenous agonist, adenosine, is an intuitively good starting point for drug design of A1R agonist
radiotracers (Figure 1b).5-6, 21 However, adenosine-like molecules are likely to have limited BBB
permeability at ng/kg to sub µg/kg concentration because of their high polar surface area and molecular
weight22-23. In fact, [18F]FNECA (8, Figure 1B), the only known adenosine-like A1R PET radioligand,
displayed very poor accumulation in the rabbit brain.21, 24 Therefore, another approach was needed to
develop a selective A1R agonist for brain imaging.
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Based on the scientific reports and patents published over the past decade,25-32 we decided to focus on two
similar non-nucleoside scaffolds (Figure 1C): 3,5-dicyanopyridine and 5-cyanopyrimidine derivatives
some of which showed desirable functional activity on A1R or A2AR. Unfortunately, the direct systematic
comparison between the two core structures has not been investigated. More importantly, these templates
have not been utilized in PET tracer development, and we envisioned that they could provide agonist lead
compounds via their structural optimization for desirable biological properties such as BBB permeability,
potency and A1R selectivity.
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O
N
O
N
O
N
H
N
A
B
H
N
NH2
N
N
N
1
2
3
4
5
6
7
8
N
N
N
N
O
N
N
N
N
O
O
O
O
n
N
N
N
18
H311C
F
N
O
1 Caffeine
2 DPCPX
3 [18F]CPFPX
Ki A1: 0.47 nM
A2A: 375 nM
Ki A1: 10,700 nM
A2A: 23,400 nM
A2B: 33,800 nM
A3: 13,300 nM
Ki A1: 3.9 nM
A2A: 129 nM
A2B: 56 nM
A3: 3,980 nM
4
[
11C]Preladenant
Ki A1: 228.4 nM
A2A: 0.38 nM
HN
NH2
NH2
N
NH2
N
N
N
N
N
N
N
9
N
N
N
N
N
H
N
10
11
12
13
14
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18
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21
22
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N
N
H
N
O
N
O
HO
HO
O
18F
O
O
O
OH OH
6 CPA
Ki A1: 2.3 nM
A2A: 794 nM
A2B: 18,600 nM
A3: 72 nM
OH OH
OH OH
7 NECA
Ki A1: 14 nM
A2A: 20 nM
A2B: 140 nM
A3: 25 nM
OH OH
8 [18F]FNECA
5 Adenosine
Ki A1: 77 nM
A2A: 0.5 nM
A3: 45 nM
R
N
OH
O
O
OH
O
C
R'
OH
O
NH
NH
O
S
NC
CN
NC
CN
S
NC
CN
S
NC
CN
H
NC
H
NC
N
N
NC
H2N
CN
S
N
H2N
S
Ar
H2N
N
N
H2N
N
H2N
N
S
S
N
H2N
N
N
S
N
H2N
N
S
Ar
N
Ar
R= H, OH, OMe
R'=H, OH, OMe
N
11 LUF 5835
10 LUF 5834
9 Capadenoson
(Bayer)
R"
Bayer patents
Otsuka patents
Leiden Univ.
late publications
Leiden Univ.
early publications
Cl
Figure 1. Representative adenosine receptor (radio)ligands and affinities at human adenosine receptors.
A). Adenosine antagonist ligands based on a xanthine or pyrazolotriazolopyrimidine scaffold. B).
Adenosine agonist ligands based on a purine nucleoside scaffold. C). Non-ribose adenosine agonist core
structures.
RESULTS AND DISCUSSION
Initially, two sets of simple methyl sulfides (Figure 2) with 3,5-dicyanopyridine and 5-cyanopyrimidine
core structures were chosen as molecular scaffolds upon which to build structures with the required BBB
permeability level for PET imaging. Keeping the thioether linker, we utilized acetamido or methoxy
substituents on the phenyl group, building on prior data that reported these functional groups to be critical
for binding potency and subtype selectivity. Both groups can be utilized as labeling positions using either
[11C]acetyl chloride or [11C]methyl iodide after structural expansion, which confers significant advantage
in the labeling processes. Calculated lipophilicity (clog P) was in the range 2-4 (shown in Figure 2), which
is considered acceptable.
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O
O
1
2
OMe
HN
OMe
HN
3
4
5
6
7
8
9
NC
NC
NC
CN
11CH3
NC
CN
11CH3
N
N
N
11CH3
11CH3
H2N
S
H2N
N
S
H2N
N
S
H2N
N
S
10
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15
clogP=3.29
14
clogP=3.09
13
clogP=2.46
12
clogP=2.19
Figure 2. Structures and time-activity curves in the whole brain of the model compounds
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Scheme 1. General synthesis of potential adenosine agonists.a
1
2
3
R
R
N
R
R
N
N
21a
21b
21c
21d
21g
R=4-OMe, Ar=
R=3-OMe, Ar=
R=4-OMe, Ar=
R=3-OMe, Ar=
R=4-NHAc, Ar=
4
5
6
7
8
9
i
ii
iii
NC
NC
N
CN
CN
Br/Cl
CHO
H
N
H2N
N
SH
Ar
H2N
N
S
Ar
16a
16b
16g
R=4-OMe
R=3-OMe
R=4-NHAc
N
N
17a
17b
17g
R=4-OMe
R=3-OMe
R=4-NHAc
H
N
18a
18b
18g
R=4-OMe
R=3-OMe
R=4-NHAc
N
10
11
12
13
14
15
16
17
18
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21
22
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iv
R
R
R
N
22a
22b
22c
22d
22g
R=4-OMe, Ar=
R=3-OMe, Ar=
R=4-OMe, Ar=
R=3-OMe, Ar=
R=4-NHAc, Ar=
N
iii
v, vi
NC
CN
Ph
NC
CN
S
NC
CN
H
N
Br/Cl
N
N
Ar
H2N
19a
19b
19g
N
S
H
N
H2N
N
Ar
H2N
N
SH
R=4-OMe
R=3-OMe
R=4-NHAc
20a
20b
20g
R=4-OMe
R=3-OMe
R=4-NHAc
N
N
N
OH
Y
OMe
Y
21e
X=N, Y=null, Ar=
21b
X=N, Y=null, Ar=
X=N, Y=null, Ar=
X=C, Y=CN, Ar=
X=C, Y=CN, Ar=
H
N
H
N
21f X=N, Y=null, Ar=
22e
21d
22b
22d
N
vii
N
N
NC
NC
H2N
N
X=C, Y=CN, Ar=
Ar 11 X=C, Y=CN, Ar=
X
X
H
N
H
N
H2N
N
S
N
S
Ar
N
N
aReagents and conditions: (i) malononitrile, piperidine, EtOH, reflux, 2 h; (ii) thiourea, K2CO3, EtOH,
reflux, 5 h; (iii) Et3N, MeCN, r.t. overnight/60 oC, 4h; (iv) malononitrile, PhSH, Et3N, EtOH, reflux, 5 h;
(v) Na2S, DMF, 80 oC; (vi) HCl (aq); (vii) 10 equiv BBr3, DCM, 0 C to r.t., overnight.
o
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Table 1. SAR of compounds 21a-g and 22a-g at adenosine receptors (human, unless noted).
2
3
4
R
R
5
6
7
8
NC
CN
S
NC
N
H2N
N
Ar
H2N
N
S
Ar
Affinity, Ki, nMa (or % inhib.)b
A1 A2A A2B
305±108 44±3%
A1
No.
R
Ar
Affinity, Ki, nMa (or % inhib.)b
A1
9
No.
R
Ar
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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32
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41
42
43
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45
46
47
48
functional
efficacy
(Emax %)c
functional
efficacy
(Emax %)c
A3
A1
A2A
A2B
A3
N
N
N
N
21a
21b
21c
21d
4-
OMe
29±0.1 32±2%
7.2±2.4 46±1%
306±17 44±0.2
48±2.8 554±273
18±2.1 93±63
-2.0±3.5
22a
22b
22c
22d
4-
OMe
2.9±0.7
35±19
30±2.5
701±244 -0.06±3.3
3-
OMe
35±10% 42±11% 3.4±1.9
3-
OMe
0.49±0.06 71±25
(0.21±0.04 (52±8.9
75±30
42±11% 16±2.3d
rat)
rat)
H
N
H
N
4-
OMe
237±48
208±27
126±12
51±5%
1.4±3.1
4.9±3.3
4-
OMe
4.6±0.6
105±37 19±3.0
20±4.6
71±13
9.4±3.2
23±3.9
N
N
N
N
H
N
H
N
3-
OMe
703±50
3-
OMe
3.1±0.4
38±5.5 38±21
N
N
21e
21f
3-OH
3-OH
79±9.4
112±14
18±3.3
41±6.1
22e
11
3-OH
3-OH
3.5±1.3
9.6±4.0
15±3.1 24±16
32±2.4 4.0±1.9
74±3.4
30±15
109±7.8
102±2.0
H
N
H
N
110±31 96.0±13.3 38±7.3
N
N
N
N
21g
4-
NHAc
59±13
3.5±0.1
9.8±4.6
147±91
117±6
22g
4-
NHAc
53±18
2.8±0.1 1.2±0.2
22±6.7
122±5.2
a Competition radioligand binding assays were conducted with membranes prepared from HEK-293 cells expressing recombinant A1, A2A, A2B
or A3Rs (human), or in rat brain membranes. The incubation was performed for 1 h at 25oC. The radioligands used were: A1R, [3H]8-cyclopentyl-
1,3-dipropylxanthine ([3H]2, 0.5 nM); A2AR, [3H]ZM241385 (0.8 nM); A2BR, [3H]5-N-ethylcarboxamidoadenosine (NECA, 7, 25 nM); A3R,
[125I]N6-(4-amino-3-iodobenzyl)adenosine-5-N-methyluronamide ([125I]I-AB-MECA, 0.1 nM). Non-specific binding was determined using 10
µM 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine (XAC, 23), 10 μM for A1 and A2A; 100 μM for A2B and
A3. Values are expressed as the mean±SEM from 2 to 4 independent experiments. b Percent inhibition at 1 µM. c cAMP accumulation assay (6
expressed as 100%). d22b has a EC50=1.0±0.2 nM at A1R.
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1
2
3
a
Scheme 2. Preparation of [11C]22b.
4
5
O11CH3
OH
6
7
8
9
i
NC
CN
S
NC
CN
S
N
N
RCY=20±7%
Purity >99%
H2N
N
H2N
N
10
11
12
13
14
15
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21
22
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[
11C]22b
22e
aReagents and conditions: (i) [11C]CH3I, tetrabutylammonium hydroxide, DMF, r.t., 3 min.
Facile S-methylation of the corresponding nor-precursors was performed using [11C]methyl iodide in high
radiochemical yield (Scheme S1, RCY= 65~90%), followed by pilot PET studies in rats (Figure 2). The
brain uptake of compounds 14 and 15 with methoxy compounds was high showing ~3 g/mL standard
uptake value (SUV) at 10 min after intravenous administration, while compounds 12 and 13 with p-
acetamido substituents showed poor BBB permeability. Since the molecular weight and lipophilicity were
within an acceptable range,33 we speculated that the polar surface area was the major factor contributing
to this difference.34
Structural diversity was expanded with a combination of substituents positioned on the phenyl group and
the thioether-linked heterocyclic group. Since the meta and para positions of the phenyl group were
reported to influence potency and selectivity,27-28, 30 we chose to derivatize these positions with hydroxyl
and methoxy groups. Two heterocycles, imidazole and 2-methylpyridine were also proposed at the
thioether linker position based on prior literature.27, 29 This allowed for a direct comparison of all the
combinations to gain a more precise insight into the structure-activity relationship (SAR).
Parallel synthesis as shown Scheme 1 was completed to generate a group of potential adenosine agonists,
and their binding affinity for A1R, A2AR, A2BR and A3R was measured, as well as the A1R functional
efficacy (Table 1). Surprisingly, the 3,5-dicyanopyridine scaffold displayed superior potency towards all
adenosine subtypes compared with their 5-cyanopyrimidine counterparts. Particularly for A1R affinity,
the 3,5-dicyanopyridine scaffold showed a ~5 to 70-fold enhancement. Similarly, overall better binding
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affinities were achieved with a 3-methoxy group compared to a 4-methoxy substituent (entries 21a-d and
22a-d). Our interest then focused on additional meta-substituted R groups, specifically a hydroxy group
(entries 21e, 21f, 22e and 11), because of the feasibility of being a precursor for corresponding 3-methoxy
substituted compound. Although not as significant as the main core scaffold, the 3-methoxy group also
favored A1R binding potency (2 to 10-fold) compared with the hydroxyl group (cf. 21b/21e, 21c/21f,
22b/22e and 22c/11), while an opposite trending was observed at the A2AR. Additionally, because of the
generally low abundance of A2BR, as well as species-dependent expression and function of the A3R35, we
would not further pursue the SAR at these two subtypes, although data was included in the comprehensive
Table 1. Moreover, it is worth noting that compounds 11, 22c and 22d possessed very similar binding
properties compared to previously reported data.27 In addition, 4-acetamido compounds 21g and 22g
displayed enhanced selectivity towards A2AR, and the high affinity of 21g was also consistent with its
previously reported EC50 at A2AR (9.0 nM).29 Therefore, our SAR studies bridged the in vitro data from
previous studies, which made the cross comparison of molecules based on the two scaffolds much easier.
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However, the functional efficacy assays revealed that a 3-hydroxyl group is favored for A1R activation
over a 3-methoxy group, indicating that the phenol group acting as a hydrogen bond donor could be
important to functional activity. There was no significant difference between the imidazole and
methylpyridine moieties at the thioether linker for A1R efficacy. Overall, considering the subtype
selectivity and functional activity, we decided to move forward with a partial agonist, 22b, which showed
subnanomolar A1R affinity at both rat and human homologues (Table 1, Supporting Information, Figures
S4 & S5).
Compound 22e, a precursor molecule for [11C]22b was prepared by demethylation of 22b using boron
tribromide (Scheme 1, yield= 62%). C-11 methylation of compound 22e was rapid including preparative
HPLC and formulation (Scheme 2, total synthesis time ~31min), providing a good radiochemical yield
(20±7%, n=6), high radiochemical purity (>99%), and high specific activity (684±259 GBq/µmol @EOB,
n=26). Measured lipophilicity was in an optimal range for BBB permeability (logD7.4 = 3.25, clogP=3.06).
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Figure 3. A). Time-activity curves for test-retest variability (n=3) in the whole rat brain. B). Regional
brain biodistribution of [11C]22b. Cd/Pu, caudate/putamen; Hipp, hippocampus; CB, cerebellum; Thal,
thalamus; Amg, amygdala; Hypothal, hypothalamus.
As shown in Figure 3, brain uptake of [11C]22b was high in cerebral cortex, hippocampus,
caudate/putamen, and cerebellum where A1R levels are high and moderate to low in brain stem where
levels are lower.36 Our initial result of three test-retest experiments (n=3) showed high reproducibility
between subjects (t-test) and between scans in the same subject in whole brain. Analytical radioHPLC
analysis of whole brain homogenate at 15 min after administration of [11C]22b indicated that the fraction
of radioactivity of parent tracer was greater than 99% (n=4, Figure S4A), which reflected that BBB
penetrable radiometabolites were negligible in brain. In plasma, about 23% [11C]22b was present at 15
min (n=2, Figure S4B), indicating a moderate metabolism.
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Figure 4. Blocking studies of [11C]22b in the rat brain. A). Time-activity curves of [11C]22b before and
after pretreatment with 2 (averaged, n=2, ip, 3.3 mg/kg). B). Averaged [11C]22b SUV images at baseline
and after pretreatment with [11C]22b and with the A2AR selective antagonist, preladenant. C). Ratio of
[11C]22b regional uptake (area under the curve, 25-80 min) before to after pretreatment. WB, whole
brain; Cd/Pu, caudate/putamen; Hipp, hippocampus; Tha, thalamus; CB, cerebellum. 22b, 1 mg/kg (n=3,
iv), 0.5 mg/kg (n=1, iv), 0.1 mg/kg (n=1, iv); 4, 3.3 mg/kg (n=3, iv); 2, 3.3 mg/kg (n=2, ip).
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For binding specificity, three rats were pretreated with iv non-radioactive 22b (1 mg/kg, n=3). As shown
in Figure 4, pretreatment with non-radioactive 22b (doses 1 and 0.5 mg/kg), or with xanthine 2 both
decreased binding of [11C]22b in all brain regions. Furthermore, blockade was larger in thalamus,
hippocampus, and striatum than midbrain and pons. Although the sample size was insufficient to assess
statistical significance, the decrease in binding appeared to be dose-dependent. We also examined binding
selectivity against A2AR (n=3), using a high dose of antagonist 4 (2 mg/kg, n=3), a selective A2AR
antagonist drug that had been in Phase 3 clinical trials. In Figure 4, no change in brain uptake was observed
with 4 pretreament, reflecting the high in vivo subtype selectivity of [11C]22b. Notably, in the caudate
and putamen, which are regions with very high A2AR levels, Preladenant pretreatment did not affect the
binding of [11C]22b. However, pretreatment with the A1 selective antagonist 2 (3.3 mg/kg, n=2) showed
an almost identical blocking profile as pretreatment with 22b alone, indicating negligible off-target
specific binding.
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CONCLUSION
The specific signal of a PET radiotracer in vivo might not be fully explained by in vitro binding assay data
due to the various functional/structural states of the target protein in vivo. Since the partial agonist
[11C]22b showed high specificity and subtype selectivity in vivo, it is a promising tool to visualize
occupancy of agonists or partial agonists in drug development. A1R partial agonists have generated
interest for their therapeutic potential to minimize side effects caused by full agonist treatments, such as
their adverse cardiac actions,37 receptor desensitization,38 sedation,39 etc. In peripheral cardiac
applications, 9, a biased partial A1R/A2BR agonist, completed Phase IIa clinical trials.40 In addition, 22b
would be a good structural motif for CNS applications due not only to its partial agonist properties, but
also its high BBB permeability. For example, a BBB penetrable A1R partial agonist might be useful for
attenuating alcohol withdrawal symptoms and for neuroprotection in neuroinflammation while
minimizing cardiac side effects.
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In summary, we developed the first highly selective partial agonist PET radioligand for brain A1R studies
based on two known non-nucleoside templates, systematically guided by PET imaging for BBB
permeability. [11C]22b could serve as a stepping stone towards the development of a full agonist
radioligand, which we predict will be more sensitive to endogenous adenosine changes. Further
evaluations are ongoing.
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EXPERIMENTAL SECTION
1.
Chemistry All solvents and most reagents were purchased from Sigma Aldrich and VWR and
used without purification unless noted. Absolute ethanol was used for HPLC eluent. 2-(Bromomethyl)-
1H-imidazole HBr salt (CAS#, 735273-40-2) was purchased from AEchem Scientific Corporation. NMR
1
spectra were recorded with a Varian 400 MHz spectrometer at 25 oC with temperature control. H and
13C NMR spectra were recorded at 400 and 100 MHz, respectively. Mass spectra were recorded on a
Advion expression S. Flash normal phase chromatography was performed on a Teledyne Isco CombiFlash
Rf system with disposable silica columns (RediSep Rf, Teledyne Isco). Analytical radio-HPLC was
performed on an Agilent 1200 series equipped with a radioflow-detector (FlowStar, Berthold; flow cell,
BGO-X 30 µL) using a Kinetex column (Phenomenex, 5µ, XB-C18 150 x 4.6 mm). GE Healthcare
TRACERlab FX-MeI and FX-M autosynthesizers were used for performing radiochemistry. The purities
of all tested compounds were determined by analytical HPLC and the purity of each compound was
confirmed ≥95% (Table S1, supporting information).
1.1 General procedures for 17a,b,g:
The synthesis was slightly modified from the literature.27 In brief, 2 drops of piperidine were added to a
solution of methoxybenzaldehyde (8 mmol) and malononitrile (1.05 equiv, 556 mg) in 6 mL EtOH. The
resulting solution was refluxed for 1.5 h until yellow solid started to precipitate out. Then, the mixture
was allowed to cool to room temperature to precipitate out more of the solid. The majority of EtOH was
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removed in vacuum and the residue was re-subjected to a mixed solvent of EtOH : hexanes = 1 : 4 (v/v).
The suspension was filtered and the filter cake was washed with more EtOH : hexanes = 1 : 4 (v/v) until
the filtrate was clear. The filter cake was dried in vacuum to get the crude product without further
purification (crude yield 89-95%).
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1.2 General procedures for 18a,b,g:
The synthesis was slightly modified from the literature.29, 31 In brief, K2CO3 (1 equiv, 138 mg) was added
to a solution of 17a,b,g (1 mmol) and thiourea (1 equiv, 76 mg) in 2 mL EtOH. The resulting mixture was
refluxed for 4 h and then allowed to cool to room temperature. Some yellow to light brown solid was
formed. The solid was filtered and washed with a mixed solvent of EtOH : hexanes = 1 : 4 (v/v). Then, it
was suspended in hot water (60 oC) and filtered. The filtrate was acidified with acetic acid to pH= 2~3 at
room temperature to precipitate out a yellow solid. The precipitate was filtered and washed with more
water, and finally dried in vacuum without further purification (crude yield 50-80%).
1.3 General procedures for 20a,b,g:
The synthesis was slightly modified from the literature.27 In brief, a drop of Et3N was added to a solution
of 17a,b,g (1 mmol), malononitrile (1 equiv, 66 mg), and thiophenol (1 equiv, 103 µL) in 2 mL EtOH.
The mixture was refluxed for 4 h to precipitate out a yellow solid. Then, the reaction mixture was cooled
to room temperature and EtOH was removed in vacuum. The residual was re-subjected to a mixed solvent
of EtOH : hexanes = 1 : 4 (v/v). The suspension was filtered and the filter cake was washed with more
EtOH : hexanes = 1 : 4 (v/v) until the filtrate was clear. The crude product 19a,b,g was dried in vacuum
without further purification (crude yield 39-63%). 19a,b,g (0.64 mmol) was then dissolved in 1.5 mL
DMF. Anhydrous Na2S was added to the resulting solution. The suspension was sonicated for 2 min and
then heated at 80 oCfor 2 h. The reaction mixture was cooled to room temperature and the majority DMF
was removed in high vacuum. 1 M HCl (aq) at room temperature was slowly added to the residual until
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Journal of Medicinal Chemistry
pH = 6. Yellow solid precipitated out and was filtered. The filter cake was washed with more water and
followed by a mixed solvent of EtOH : hexanes = 1 : 4 (v/v). The crude product 20a,b,g was dried in
vacuum without further purification (crude yield 87-99%).
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1.4 General procedures for 12-15, 21a-d,g and 22a-d,g:
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To a suspension of 18a,b,g/20a,b,g and corresponding arylbromide/arylchloride/methyliodide in ACN
was added Et3N (2 equiv), and the mixture turned to darker solution. The resulting solution was allowed
to stir at room temperature overnight and dried in vacuum. The residual was purified by flash silica
chromatography (0-8% MeOH in CHCl3 gradient with 0.1% NH4OH).
1.5 General procedures for 21e, 21f, 22e and 11:
To a suspension of corresponding methoxy compounds (21b, 22b, 21d, 22d) in anhydrous
dichloromethane was added BBr3 (1.0 M in DCM, 10 equiv) dropwise at 0 oC. The reaction mixture
was allowed to slowly warm to room temperature and stirred overnight. The resulting
dark brown suspension was diluted with more DCM and quenched by a few drops of
iPrOH at 0 oC. The mixture turned orange, and more saturated NaHCO3 and DCM were
added. The yellow colored organic layer was separated and the aqueous layer was
extracted with DCM three times. The combined organic layers were dried with Na2SO4
and purified by flash silica chromatography (0-10% MeOH in CHCl3 gradient with 0.1% NH4OH).
NMR spectra for new compounds can be found in supporting information.
N-(4-(2-Amino-3,5-dicyano-6-(methylthio)pyridin-4-yl)phenyl)acetamide (12). Pale yellow solid (20
mg, 96% yield). 1H NMR (d6-DMSO, 400 MHz): 10.19 (s, 1H), 7.95 (broad, s, 2H), 7.74 (d, J = 8.6 Hz,
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2H), 7.46 (d, J = 8.6 Hz, 2H), 2.58 (s, 3H), 2.09 (s, 3H). 13C NMR (d6-DMSO, 100 MHz): 169.2, 167.9,
160.2, 158.2, 141.6, 129.6, 128.5, 119.1, 116.0, 115.9, 90.1, 85.7, 24.5, 13.2. MS (ESI+): m/z calcd for
C16H14N5OS [M+H]+ 324.09, found 324.13
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N-(4-(6-Amino-5-cyano-2-(methylthio)pyrimidin-4-yl)phenyl)acetamide (13). Yellow solid (14 mg,
67% yield). 1H NMR (d6-DMSO, 400 MHz): 10.21 (s, 1H), 7.87 (d, J = 8.4 Hz), 7.80 (broad, s, 2H), 7.73
(d, J = 8.4 Hz), 2.50 (s, 3H), 2.09 (s, 3H). 13C NMR (d6-DMSO, 100 MHz): 174.1, 169.2, 166.8, 163.9,
142.4, 130.6, 129.8, 118.8, 117.0, 81.2, 24.6, 13.9. MS (ESI+): m/z calcd for C14H14N5OS [M+H]+
300.09, found 300.16
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2-Amino-4-(4-methoxyphenyl)-6-(methylthio)pyridine-3,5-dicarbonitrile (14). Light yellow solid
(12 mg, 52% yield). 1H NMR (CDCl3/d4-MeOD=5/1, v:v, 400 MHz): 7.43 (d, J = 8.8 Hz, 2H), 6.99 (d, J
= 8.8 Hz, 2H), 3.82 (s, 3H), 2.54 (s, 3H). 13C NMR (CDCl3/d4-MeOD=5/1, v:v, 100 MHz): MS (ESI+):
169.4, 161.5, 159.8, 157.8, 130.1, 125.5, 115.7, 115.4, 114.2, 95.3, 85.6, 55.2, 13.2. MS (ESI+): m/z calcd
for C15H13N4OS [M+H]+ 297.08, found 297.12
4-Amino-6-(4-methoxyphenyl)-2-(methylthio)pyrimidine-5-carbonitrile (15). White solid (11 mg,
40% yield). 1H NMR (d6-DMSO, 400 MHz): 7.90 (d, J = 8.4 Hz, 2H), 7.79 (broad, s, 2H),7.10 (d, J = 8.4
Hz, 2H), 3.85 (s, 3H), 2.51 (s, 3H). 13C NMR (d6-DMSO, 100 MHz): 174.0, 166.7, 163.8, 162.0, 130.8,
129.5, 117.1, 114.3, 81.7, 55.9, 13.9. MS (ESI+): m/z calcd for C13H13N4OS [M+H]+ 273.08, found
273.11
4-Amino-6-(4-methoxyphenyl)-2-(((6-methylpyridin-2-yl)methyl)thio)pyrimidine-5-carbonitrile
(21a). White solid (14 mg, 50 % yield). 1H NMR (CDCl3, 400 MHz): 8.02 (d, J = 8.9 Hz, 2H), 7.53-7.47
(m, 1H), 7.28-7.24 (m, 1H), 7.04-7.01 (m, 1H), 6.99 (d, J = 8.9 Hz, 2H), 5.86 (broad, s, 2H), 4.54 (s, 2H),
3.87 (s, 3H), 2.56 (s, 3H). 13C NMR (CDCl3, 100 MHz): 173.9, 166.3, 163.7, 162.4, 158.0, 156.7, 136.9,
130.6, 127.9, 121.7, 119.9, 116.8, 114.0, 82.2, 55.4, 37.1, 24.3. MS (ESI+): m/z calcd for C19H18N5OS
[M+H]+ 364.12, found 364.19
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4-Amino-6-(3-methoxyphenyl)-2-(((6-methylpyridin-2-yl)methyl)thio)pyrimidine-5-carbonitrile
(21b) Yellow solid (26 mg, 72 % yield). 1H NMR (CDCl3, 400 MHz): 7.59-7.55 (m, 1H), 7.54-7.47 (m,
2H), 7.43-7.38 (m, 1H), 7.29-7.25 (m, 1H), 7.10-7.06 (m, 1H), 7.06-7.02 (m, 1H), 5.84 (broad, s, 2H),
4.55 (s, 2H), 3.86 (s, 3H), 2.56 (s, 3H). 13C NMR (CDCl3, 100 MHz): 174.3, 167.1, 163.5, 159.6, 158.1,
156.5, 137.0, 136.8, 129.7, 121.8, 121.1, 119.9, 117.7, 116.2, 113.7, 83.4, 55.4, 37.2, 24.3. MS (ESI+):
m/z calcd for C19H18N5OS [M+H]+ 364.12, found 364.17
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2-(((1H-Imidazol-2-yl)methyl)thio)-4-amino-6-(4-methoxyphenyl)pyrimidine-5-carbonitrile (21c).
Creamy white solid (52 mg, 65%). 1H NMR (d6-DMSO, 400 MHz): 11.84 (broad, s, 1H), 8.01 (broad, s,
2H), 7.90 (d, J = 8.5 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.94 (s, 2H), 4.42 (s, 2H), 3.84 (s, 3H). 13C NMR
(d6-DMSO, 100 MHz): 172.8, 166.9, 164.0, 163.9, 162.1, 143.4, 130.8, 128.4, 117.0, 114.3, 82.0, 55.9,
28.0. MS (ESI+): m/z calcd for C16H15N6OS [M+H]+ 339.10, found 339.12
2-(((1H-Imidazol-2-yl)methyl)thio)-4-amino-6-(3-methoxyphenyl)pyrimidine-5-carbonitrile (21d).
White solid (55 mg, 71%). 1H NMR (d6-DMSO, 400 MHz): 8.02 (broad, s, 2H), 7.49-7.36 (m, 3H), 7.18-
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7.11 (m, 1H), 6.94 (s, 2H), 4.43 (s, 2H), 3.82 (s, 3H). C NMR (d6-DMSO, 100 MHz): 173.0, 167.6,
163.8, 159.5, 143.4, 137.6, 130.1, 121.2, 117.3, 116.6, 114.3, 110.0, 83.2, 55.8, 28.0. MS (ESI+): m/z
calcd for C16H15N6OS [M+H]+ 339.10, found 339.13
4-Amino-6-(3-hydroxyphenyl)-2-(((6-methylpyridin-2-yl)methyl)thio)pyrimidine-5-carbonitrile
1
(21e). Pale yellow solid (56 mg, 72%). H NMR (CDCl3/d4-MeOD=5/1, v:v, 400 MHz): 7.57-7.51 (m,
1H), 7.42 (broad, s, 1H), 7.36-7.28 (m, 3H), 7.28-7.23 (m, 1H), 7.07-7.02 (m, 1H), 6.98-6.93 (m, 1H),
4.45 (s, 2H), 2.50 (s, 3H). 13C NMR (CDCl3/d4-MeOD=5/1, v:v, 100 MHz): 173.5, 167.7, 163.7, 157.6,
157.0, 156.7, 137.6, 136.8, 129.6, 122.2, 120.5, 119.8, 118.5, 116.2, 115.3, 82.9, 36.3, 23.3. MS (ESI+):
m/z calcd for C18H16N5OS [M+H]+ 350.11, found 350.11
2-(((1H-Imidazol-2-yl)methyl)thio)-4-amino-6-(3-hydroxyphenyl)pyrimidine-5- carbonitrile (21f).
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White solid (16 mg, 54%). H NMR (d6-DMSO, 400 MHz): 11.83 (broad, s, 1H), 9.79 (s, 1H), 8.21
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13
(broad, s, 1H), 7.79 (broad, s, 1H), 7.35-7.24 (m, 3H), 7.04-6.85 (m, 3H), 4.42 (s, 2H). C NMR (d6-
DMSO, 100 MHz): 173.2, 168.0, 164.1, 157.9, 143.5, 137.7, 130.2, 119.8, 118.7, 116.8, 115.8, 83.1, 29.2.
MS (ESI+): m/z calcd for C15H13N6OS [M+H]+ 325.09, found 325.13
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2-Amino-4-(4-methoxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile
(22a). Yellow solid (80 mg, 97 % yield). 1H NMR (CDCl3, 400 MHz): 7.59-7.53 (m, 1H), 7.50 (d, J =
8.8 Hz, 2H), 7.26-7.22 (m, 1H), 7.10-7.05 (m, 1H), 7.04 (d, J = 8.8 Hz, 2H), 5.74 (broad, s, 2H), 4.58 (s,
2H), 3.87 (s, 3H), 2.58 (s, 3H). 13C NMR (CDCl3, 100 MHz): 168.3, 162.5, 161.7, 159.3, 157.8, 155.4,
137.1, 130.2, 125.2, 122.1, 120.0, 115.6, 115.0, 114.4, 96.3, 86.7, 55.4, 36.7, 24.3. MS (ESI+): m/z calcd
for C21H18N5OS [M+H]+ 388.12, found 388.20
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(22b). Pale yellow solid (17 mg, 62%). H NMR (CDCl3/d4-MeOD=5/1, v:v, 400 MHz): 7.59-7.52 (m,
1H), 7.40-7.33 (m, 1H), 7.33-7.26 (m, 1H), 7.08-7.02 (m, 1H), 7.02-6.96 (m, 2H), 6.95-6.90 (m, 1H), 4.50
(s, 2H), 3.79 (s, 3H), 2.51 (s, 3H). 13C NMR (CDCl3/d4-MeOD=5/1, v:v, 100 MHz): 167.2, 159.8, 159.6,
158.2, 157.6, 156.1, 137.8, 134.5, 130.0, 122.4, 120.6, 120.5, 116.6, 115.2, 114.9, 113.6, 94.8, 86.3, 55.3,
35.4, 23.5. MS (ESI+): m/z calcd for C21H18N5OS [M+H]+ 388.12, found 388.18
2-Amino-4-(3-hydroxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile
(22e). Light yellow solid (38 mg, 62%). 1H NMR (CDCl3/d4-MeOD=5/1, v:v, 400 MHz): 7.60-7.52 (m,
1H), 7.32-7.24 (m, 2H), 7.09-7.02 (m, 1H), 6.96-6.90 (m, 1H), 6.90-6.83 (m, 2H), 4.50 (s, 2H), 2.51 (s,
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3H). C NMR (CDCl3/d4-MeOD=5/1, v:v, 100 MHz): 167.2, 159.8, 158.5, 157.7, 157.1, 156.1, 137.7,
134.5, 130.0, 122.4, 120.6, 119.4, 117.8, 115.3, 115.1, 115.0, 94.7, 86.3, 35.4, 23.5. MS (ESI+): m/z calcd
for C20H16N5OS [M+H]+ 374.11, found 374.12
N-(4-(2-Amino-3,5-dicyano-6-(((6-methylpyridin-2-yl)methyl)thio)pyridin-4-yl)phenyl)acetamide
(22g). Yellow solid (25 mg, 78%). 1H NMR (CDCl3/d4-MeOD=5/1, v:v, 400 MHz): 7.72-7.64 (m, 2H),
7.59-7.53 (m, 1H), 7.42-7.36 (m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 4.50 (s, 2H),
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2.50 (s, 3H), 2.11 (s, 3H). 13C NMR (CDCl3/d4-MeOD=5/1, v:v, 100 MHz): 170.0, 167.3, 159.9, 157.9,
157.7, 156.1, 140.9, 137.8, 129.2, 128.3, 122.4, 120.6, 119.5, 115.4, 115.2, 94.6, 86.2, 35.4, 23.7, 23.5.
MS (ESI+): m/z calcd for C22H19N6OS [M+H]+ 415.13, found 415.20.
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Radiochemistry No-carrier-added (n.c.a.) [11C]CO2 was generated by bombarding a N2/1% O2
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gas target with a proton beam (16 MeV, 45 μA × 20 min) from a cyclotron (PETrace, GE) via nuclear
reaction 14N(p, α)11C. Radiochemistry including conversion from [11C]CO2 to [11C]CH3I and
[11C]methylation was performed in a lead-shielded hot cell on FX-MeI and FX-M automated synthesizers
(GE Healthcare, USA), respectively, controlled by TRACERlab software.
2.1 Preparation of [11C]12-15 and 22b:
A solution of precursor 22e (20g, 18g, 20a and 18a for 12-15, respectively) (0.50 mg ± 0.05 mg) and 3
μL tetrabutylammonium hydroxide (1M in methanol) in DMF (80 μL) was vortexed and loaded into an
HPLC injection loop (2 mL capacity)41-42. The mixture was allowed to react with [11C]CH3I in a stream
of helium at ambient temperature for ~5 min before being injected to a semi-preparative HPLC column
(Phenomenex, Gemini-NX, 5μ, C18, 250 × 10 mm). The mixture was eluted at 5 mL/min with an isocratic
mixture of 55% solvent A (95% H2O, 5% EtOH, 0.01 M HCl, pH=2.1) and 45% solvent B (40% H2O,
60% EtOH, 0.01 M HCl, pH=2.1) and monitored for absorbance at 254 nm and radioactivity using the
flow count detector (NaI(Tl)) built into the FX-M. The product [11C]22b was collected between 11.2 to
11.9 min (Figure S1) and radioactivity was measured by a dose calibrator (Capintec, CRC 712M) to
determine radiochemical yield (20 ± 7%, n=6) and specific activity (684 ± 259 GBq/µmol @EOB, n=26).
The collected product solution was formulated with 4 mL 0.1 M sodium phosphate buffer (pH= ~6) for
rodent PET studies.
Radiosynthesis data and characterization of [11C]22b can be found in supporting information.
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3.
Log D7.4 measurement Lipophilicity was determined by using a method for log D7.4 found in
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previous literature.43 A mixture of 2.5 mL 1-octanol and 2.5 mL 0.1 M phosphate buffer (pH = 7.4) was
spiked with approximately 3.7 MBq [11C]22b. The mixture was then vortexed for 2 min and centrifuged
for 2 min at 7000 rpm to fully separate the two layers. Aliquots from both layers (0.1 mL octanol layer, 1
mL aqueous layer) were counted for 2 min using a gamma counter (Packard, COBRA II auto-gamma
counter). A majority of the octanol layer (2 mL) was then transferred to a mixture of fresh octanol (0.5
mL) and phosphate buffer (2.5 mL). This procedure was repeated 10 times to obtain a stable value for log
D7.4. The mean log D7.4 of [11C]22b was 3.249 (n = 5, SD = 0.022).
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Small animal PET studies All animal studies were approved by the Clinical Center Animal Care
and Use Committee of National Institutes of Health (Protocol number, NIAAA-15-01). Studies were
performed on male Wistar rats (2-3 month old, Charles River Laboratories, 318 40 g) in a small animal
PET scanner (Siemens, microPET Focus 220). Rats were anesthetized using 2-5% isoflurane
approximately 1 hr before the first scan. Body temperature was measured and controlled throughout the
study using a heating blanket and rectal probe (36.9  0.4 C, Harvard Apparatus Homeothermic Blanket
System). Heart rate and oxygen saturation were monitored using a pulse oximeter (Kent Scientific
MouseSTAT Jr.).
After obtaining a 10 min transmission scan, rats were injected with [11C]22b formulated in saline (dose,
20.837.0 MBq; mean injected volume, 448 137 L; specific activity, 155  55 GBq/mol at time of
injection). [11C]22b was administered intravenously through either a tail vein or penile catheter using a
syringe pump (rate, 600 L/min; Harvard Apparatus). Following injection, approximately 100 L of
heparinized saline was used to wash out residual radioactivity in the catheter. Dynamic PET scans were
performed in list mode for 90 min and image reconstruction was done by the software associated with the
scanner (MicroPET Manager). PMOD software (ver 3.8, PMOD Technologies) was used for
coregistration to a template44 and then used to generate a time-activity curve for each ROI.
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This procedure was duplicated for test/retest (n = 3) on the same study day 30 min after the first scan was
completed. For blocking studies, the pretreatment drug was administered 10 min before the second
injection of [11C]22b through either the same injection site as that of the radiotracer or through an
intraperitoneal injection. The pretreatment drugs used were Preladenant(4) (2.0 mg/kg, IV, n = 3) for
testing binding specificity to A2AR, 22b (1.0 mg/kg, IV, n = 3) for testing specific binding, and 2 (2 mg/kg,
IP, n = 2) for testing binding specificity to A1R.
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RadioHPLC analyses on rat brain homogenate and plasma.
[11C]22b (injection dose, 2-5 mCi) were injected to rat (Wistar, 250-350g, n=4) intravenously under
anesthesia. At 15 min after [11C]22b administration, blood samples (1-2 mL) were obtained through
cardiac puncture and rats were immediately decapitated and brains were harvested. Whole blood samples
were centrifuged 14,500 RPM for two minunites and the resulting plasma samples were treated with
acetonitrile to remove plasma proteins. Brain samples were treated with acetonitrile (2 mL) and
homogenized by homogenizer (099C K5424, Glas-Col, IN) at 3,000 RPM for four minunites. Both brain
and plasma samples were centrifuged and filtered to give the supernatant solutions for radioHPLC
analyses.
RadioHPLC analyses on brain homogenate and plasma were performed on Agilent 1260 system using
UV detector at 254 nm and a flow count radioactivity detector (Berthold LB 514 with BGO-X 30 µL flow
cell). Phenomenex Luna 10µ C18(2) column (250 × 4.6 mm), solvent A (H2O (0.05M ammonia formate
and 0.5% HOAc)) 25%, solvent B (acetonitrile) 75%, flow rate: 1 mL/min. Cold 22b was spiked in
samples as internal standard.
6.
Radioligand binding assays
6.1 Materials [3H]2 (6.1 GBq/µmol), [3H]7 (1.1 GBq/µmol), [125I]AB-MECA (74 GBq/µmol), and
AlphaScreen cAMP kits were purchased from PerkinElmer (Waltham, MA, USA). [3H]ZM241385 (1.8
GBq/µmol) was from American Radiolabeled Chem. Inc (St. Louis, MO, USA). 8-[4-[4-(4-
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Chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB603) was purchased from Tocris
(Minneapolis, MN, USA).
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6.2 Cell culture and membrane preparation HEK293 cells expressing the recombinant human ARs
were grown in DMEM supplemented with 10% FBS at 37°C in a humidified incubator containing 5%
CO2. Cells were collected after confluence by scraping into ice-cold PBS buffer. After homogenization
and suspension, cells were centrifuged at 1,000g for 10 min, and the suspension was recentrifuged at
20,000g for 60 min at 4°C. The resultant pellets were resuspended in buffer containing 3 units/mL
adenosine deaminase (Worthington Biochemical Corp., Lakewood, NJ) and incubated at 37°C for 30 min.
The membrane preparation was split into aliquots which were kept at −80 °C until further use.
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6.3 Competative radioligand binding assay HEK293 cell membrane homogenates (20 μg protein/tube
for A1R, A2AR, and A3R; 50 μg protein/tube for A2BR) or rat brain homogenates (forebrain for A1R and
striatum for A2AR) were incubated at 25ºC for 60 min with various concentrations of test compounds and
radioligands ([3H]2 (0.5 nM) for A1R; [3H]ZM241385 (0.8 nM) for A2AR); [3H]7 (25 nM) for A2BR;45-46
[125I]AB-MECA (0.1 nM) for A3R) in 50 mM Tris.HCl buffer containing 10 mM MgCl2 Nonspecific
binding was determined in the presence of 23 (10 μM for A1 and A2A; 100 μM for A2B and A3). Binding
reaction was terminated by rapid filtration through Whatman GF/B filters using a MT-24 cell harvester
(Brandel, Gaithersburg, MD, USA). Filters were washed 3 times with 3 mL of ice-cold buffer. For the
binding of [3H]2, [3H]ZM241385, and [3H]7, radioactivity was determined using liquid scintillation
counter (Tri-Carb 2810TR; PerkinElmer). For the binding of [125I]AB-MECA, radioactivity was analyzed
using a PerkinElmer Cobra II γ-counter. Binding parameters were calculated using Prism 7 software
(GraphPAD, San Diego, CA, USA). IC50 values were converted to Ki values using the Cheng–Prusoff
equation.47 The Ki values from binding experiments are expressed as mean ± standard error from 2-4
independent measurements performed in duplicate.
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6.4 cAMP accumulation assay Inhibition of forskolin-stimulated cAMP accumulation in HEK293 cells
expressing the A1R was performed as described previously.48 Cells were seeded into 384-well culture
plates pre-coated with poly-D-lysine at a density of 1,000 cells/well and incubated overnight at 37°C in
5% CO2 prior to assay. Cells were first incubated with 3 units/ml adenosine deaminase, rolipram (10 µM),
and PSB603 (1 µM; to antagonize endogenously expressed A2BR for 30 min at 37°C.49 Cells were then
exposed to agonists for 20 min and followed by addition of forskolin (10 μM) and incubated for another
15 min at 37°C. Reaction was terminated by removal of reaction buffer and addition of 25 μL lysis buffer
(0.3% Tween-20). Detection of cAMP was performed using AlphaScreen methods following the
manufacturer’s instructions.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
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Experimental description of the synthesis, H and 13C NMR and MS data of new compounds.
Radiosynthesis data of PET ligands [11C]22b. Experimental protocols for PET studies on rodents.
Procedures and additional data for in vitro evaluation of compounds 21a-g, 22a-g and 11. (PDF)
Molecular formula strings of new compounds. (CSV)
AUTHOR INFORMATION
Corresponding Authors
* Email: nvolkow@nida.nih.gov. Tel: +1 (301) 443-6480,
Sunny.kim@nih.gov Tel +1 (301) 496-5548.
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ORCID
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Min Guo: 0000-0003-4632-7116
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Kenneth A. Jacobson: 0000-0001-8104-1493
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
Funding Sources
This research is supported by the National Institutes of Health Intramural Research Program (Y1AA-
3009, Nora D. Volkow; ZIADK031117, Kenneth A. Jacobson)
Notes
The authors declare no potential conflict of interest.
ACKNOWLEDGMENT
Authors would like to thank NIAAA and NIDDK Intramural Research Program.
ABBREVIATIONS
AR, adenosine receptor; CNS, central neural system; BBB, blood-brain barrier; PET, positron emission
tomography
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