Preclinical evaluation of the first adenosine A1 receptor partial agonist

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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

Preclinical evaluation of the first adenosine A1 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†,*, 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 5cyanopyrimidine) 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 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 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-(2phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone ([11C]FR194921) showed acceptable bloodbrain 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. ACS Paragon Plus Environment

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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. 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.



O

O

A

N

N O

N

N

O

1 Caffeine Ki A1: 10,700 nM A2A: 23,400 nM A2B: 33,800 nM A3: 13,300 nM

O

H N

N

N

N

H N

N O

NH2

18 F [18F]CPFPX

2 DPCPX Ki A1: 3.9 nM A2A: 129 nM A2B: 56 nM A3: 3,980 nM

N N

N

N

N

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O N

H311C O

3 Ki A1: 0.47 nM A2A: 375 nM

n

N

N N

N

O

4 [11C]Preladenant Ki A1: 228.4 nM A2A: 0.38 nM

B HN

NH2 N N HO

N

N

N

N HO

O

N

H N

O

O

OH OH

OH OH

6 CPA Ki A1: 2.3 nM A2A: 794 nM A2B: 18,600 nM A3: 72 nM

5 Adenosine Ki A1: 77 nM A2A: 0.5 nM A3: 45 nM

C

NH2 N

N

O

18F

O

R

S

Bayer patents

N

Ar

S

H 2N

9 Capadenoson (Bayer)

H 2N

N S

Otsuka patents Cl

CN

NC NC

N

OH

O O

NC

S

OH

R'

CN N

N N

O

OH OH

NC H 2N

O

N

8 [18F]FNECA

O

CN N

H N

NH

NC

H 2N

N

7 NECA Ki A1: 14 nM A2A: 20 nM A2B: 140 nM A3: 25 nM

NH

NC

N

OH OH

OH

O

NH2 N

N

Ar

N

S

N

Ar

R= H, OH, OMe R'=H, OH, OMe Leiden Univ. early publications

H 2N

N

H 2N

S N

Leiden Univ. late publications

NC

CN N

H N

S

H 2N

CN N

N

N

S 10 LUF 5834

H N

S

11 LUF 5835

R"

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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Journal of Medicinal Chemistry O

O HN

HN

NC

CN 11

H 2N

N

S

CH3

12 clogP=2.19

NC

NC

N 11

H 2N

N

OMe

OMe

S

CH3

13 clogP=2.46

CN 11

H 2N

N

S

CH3

14 clogP=3.09

NC

N 11

H 2N

N

S

CH3

15 clogP=3.29

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 R

R

R i

ii

NC

CN

CHO 16a R=4-OMe 16b R=3-OMe 16g R=4-NHAc

17a R=4-OMe 17b R=3-OMe 17g R=4-NHAc

SH Ar

N

N

21a R=4-OMe, Ar=

iii

N

H 2N

CN

R

NC Br/Cl

N

H 2N

S

N

N

21b R=3-OMe, Ar= Ar

H N

21c R=4-OMe, Ar= N


H N

21d R=3-OMe, Ar= N

N

21g R=4-NHAc, Ar=

iv R

R

R

N

22a R=4-OMe, Ar= NC H 2N

CN S

N

iii

v, vi

NC

Ph

H 2N


21d X=N, Y=null, Ar=

N

SH

Br/Cl Ar

N N

H 2N

N

S

H N

22d X=C, Y=CN, Ar=

H 2N

X N

vii

S

Ar

H N

22d R=3-OMe, Ar= 22g R=4-NHAc, Ar=

OH

Y

Ar

N

N

OMe

NC

H N

22c R=4-OMe, Ar=

21f X=N, Y=null, Ar= NC H 2N

X N

Y S

H N N N

22e X=C, Y=CN, Ar= Ar

N

N

21e X=N, Y=null, Ar=

H N

22b X=C, Y=CN, Ar=

N

22b R=3-OMe, Ar=

CN


N

21b X=N, Y=null, Ar=

NC

CN

H N

11 X=C, Y=CN, 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 oC to r.t., overnight.


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Table 1. SAR of compounds 21a-g and 22a-g at adenosine receptors (human, unless noted). R

R

NC H2N

No.

NC

N N

S

H2N

Ar

R

Ar

Affinity, Ki, nMa (or % inhib.)b A1

A2A

A2B

A3

A1 functional efficacy (Emax %)c

No.

CN N

S

R

Ar

Ar

Affinity, Ki, nMa (or % inhib.)b A1

A2A

A2B

A3

A1 functional efficacy (Emax %)c

21a

4OMe

N

29±0.1

32±2%

305±108

44±3%

-2.0±3.5

22a

4OMe

N

2.9±0.7

35±19

30±2.5

701±244

-0.06±3.3

21b

3OMe

N

7.2±2.4

46±1%

35±10%

42±11%

3.4±1.9

22b

3OMe

N

42±11%

16±2.3d

4OMe

306±17

44±0.2

237±48

51±5%

1.4±3.1

22c

4OMe

H N

71±25 (52±8.9 rat) 105±37

75±30

21c

0.49±0.06 (0.21±0.04 rat) 4.6±0.6

19±3.0

20±4.6

9.4±3.2

H N

3.1±0.4

38±5.5

38±21

71±13

23±3.9

3.5±1.3

15±3.1

24±16

74±3.4

109±7.8

9.6±4.0

32±2.4

4.0±1.9

30±15

102±2.0

53±18

2.8±0.1

1.2±0.2

22±6.7

122±5.2

21d

3OMe

21e

3-OH

21f

3-OH

H N N H N

48±2.8

554±273

208±27

703±50

4.9±3.3

22d

N N

H N

3OMe

18±2.1

93±63

126±12

79±9.4

18±3.3

22e

3-OH

110±31

96.0±13.3

38±7.3

112±14

41±6.1

11

3-OH

N

21g

4NHAc

N

N

N N

H N N

59±13

3.5±0.1

9.8±4.6

147±91

117±6

22g

4NHAc

a Competition

N

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-cyclopentyl1,3-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. ACS Paragon Plus Environment


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Scheme 2. Preparation of [11C]22b.a O11CH3

OH

NC H 2N

N 22e

aReagents

i

CN S

N

RCY=20±7% Purity >99%

NC H 2N

CN N

S

N

[11C]22b

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 pacetamido 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. 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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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.

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. ACS Paragon Plus Environment

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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.

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 spectra were recorded with a Varian 400 MHz spectrometer at 25 oC with temperature control. 1H 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%). 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 oC for 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 ACS Paragon Plus Environment

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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%). 1.4 General procedures for 12-15, 21a-d,g and 22a-d,g: 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, ACS Paragon Plus Environment


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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 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 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 ACS Paragon Plus Environment

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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 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), 7.187.11 (m, 1H), 6.94 (s, 2H), 4.43 (s, 2H), 3.82 (s, 3H). 13C 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 (21e). Pale yellow solid (56 mg, 72%). 1H 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). White solid (16 mg, 54%). 1H NMR (d6-DMSO, 400 MHz): 11.83 (broad, s, 1H), 9.79 (s, 1H), 8.21 ACS Paragon Plus Environment


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(broad, s, 1H), 7.79 (broad, s, 1H), 7.35-7.24 (m, 3H), 7.04-6.85 (m, 3H), 4.42 (s, 2H). 13C NMR (d6DMSO, 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 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 2-Amino-4-(3-methoxyphenyl)-6-(((6-methylpyridin-2-yl)methyl)thio)pyridine-3,5-dicarbonitrile (22b). Pale yellow solid (17 mg, 62%). 1H 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, 3H). 13C 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), ACS Paragon Plus Environment

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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. 2.

Radiochemistry No-carrier-added (n.c.a.) [11C]CO2 was generated by bombarding a N2/1% O2

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.



3.

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Log D7.4 measurement Lipophilicity was determined by using a method for log D7.4 found in

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). 4.

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. 5.

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). 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. 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.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at

Experimental description of the synthesis, 1H 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: [email protected]. Tel: +1 (301) 443-6480, [email protected] Tel +1 (301) 496-5548.



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ORCID Min Guo: 0000-0003-4632-7116 Kenneth A. Jacobson: 0000-0001-8104-1493 Author Contributions 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 (Y1AA3009, 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 REFERENCES

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Preclinical evaluation of the first adenosine A1 receptor partial agonist

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