Design, synthesis and biological evaluation of novel scaffold benzo[4,5]imidazo [1,2-a]pyrazin-1-amine: Towards adenosine A2A receptor (A2A AR) antagonist

Article abstract of DOI:10.1016/j.ejmech.2020.113040

Antagonists of adenosine receptor are under exploration as potential drug candidates for treatment of neurological disorders, depression, certain cancers and potentially used as a cancer immunotherapy. Herein, we describe design and synthesis of novel scaffold benzo[4,5]imidazo [1,2-a]pyrazin-1-amine (6) derivatives. All the compounds were evaluated for A_2A AR antagonist activity and displayed encouraging results (IC₅₀ 9–300 nM) of A_2A AR antagonist binding affinity in biochemical assay. Compound 27 exhibits good activity in A_2A AR antagonist cAMP functional assay (IC₅₀ 31 nM) and further this compound shows T-cell activation at the IL-2 production assay (EC₅₀ 165 nM). Molecular docking studies were carried out to rationalize the observed binding affinity of compound 27.

Full text of DOI:10.1016/j.ejmech.2020.113040

Journal Pre-proof
Design, synthesis and biological evaluation of novel scaffold benzo[4,5]imidazo
[1,2-a]pyrazin-1-amine: towards adenosine A receptor (A AR) antagonist
2A
2A
G. Lakshma Reddy, Rupam Sarma, Shuhao Liu, Weifeng Huang, Jinping Lei,
Jiasheng Fu, Wenhao Hu
PII:
S0223-5234(20)31012-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.113040
EJMECH 113040
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2020
Revised Date: 11 November 2020
Accepted Date: 14 November 2020
Please cite this article as: G.L. Reddy, R. Sarma, S. Liu, W. Huang, J. Lei, J. Fu, W. Hu, Design,
synthesis and biological evaluation of novel scaffold benzo[4,5]imidazo [1,2-a]pyrazin-1-amine: towards
adenosine A receptor (A AR) antagonist, European Journal of Medicinal Chemistry, https://
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Graphical Abstract
Design, synthesis and biological evaluation of novel scaffold benzo[4,5]imidazo[1,2-
a]pyrazin-1-amine: towards adenosine A2A receptor (A2A AR) antagonist
G. Lakshma Reddy, Rupam Sarma, Shuhao Liu, Weifeng Huang, Jinping Lei,* Jiasheng Fu,*
Wenhao Hu*
NH2
N
N
N
A2A IC50= 9.2 nM
A1
IC50 = 733 nM
cAMP IC50 = 31 nM
IL-2 EC50 = 165 nM

Design, synthesis and biological evaluation of novel scaffold benzo[4,5]imidazo
[1,2-a]pyrazin-1-amine: towards adenosine A2A receptor (A2A AR) antagonist
G. Lakshma Reddy,a# Rupam Sarma,a,b# Shuhao Liu,a Weifeng Huang,a Jinping Lei,*,a Jiasheng
Fu,*,a Wenhao Hu*,a
a Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical
Sciences, Sun Yat-Sen University, Guangzhou 510006, China
bDepartment of Chemistry, Nalbari College, Assam 781335, India.
#Authors contributed equally to this work
*Corresponding author
E-mail: huwh9@mail.sysu.edu.cn
1

Abstract:
Antagonists of adenosine receptor are under exploration as potential drug candidates for
treatment of neurological disorders, depression, certain cancers and potentially used as a cancer
immunotherapy. Herein, we describe design and synthesis of novel scaffold benzo[4,5]imidazo
[1,2-a]pyrazin-1-amine (6) derivatives. All the compounds were evaluated for A2A AR antagonist
activity and displayed encouraging results (IC50 9 to 300 nM) of A2A AR antagonist binding
affinity in biochemical assay. Compound 27 exhibits good activity in A2A AR antagonist cAMP
functional assay (IC50 31 nM) and further this compound shows T-cell activation at the IL-2
production assay (EC50 165 nM). Molecular docking studies were carried out to rationalize the
observed binding affinity of compound 27.
Keywords: Benzo[4,5]imidazo[1,2-a]pyrazin-1-amine, Adenosine A2A receptor (A2A AR)
antagonist, T-Cell activation, cancer immunotherapy.
2

1. Introduction
Adenosine, a naturally occurring nucleoside is present in all tissues of mammalian organisms.
Extracellular adenosine modulates a wide range of physiological processes through interacting
with cell surface adenosine receptors (ARs) which belong to a super family of G protein coupled
receptors (GPCRs) having seven transmembrane helices.[1] There are four subtypes of AR (A1,
A2A, A2B, and A3) which differ in size (A1, A2B, A3 and A2A consist of 326, 328, 318 and 409
amino acids, respectively), exhibit unique tissue distributions.[2] While A1, A2A, and A3
receptors show high affinity, A2B receptor has low affinity for adenosine binding.[3] Among
these A2A and A2B, ARs increase the intracellular second messenger 3′,5′-cyclic adenosine
monophosphate (cAMP) levels by coupling to Gs (A2A), Gs /Gq (A2B), whereas the A1 and A3 AR
subtypes decreases intracellular cAMP levels via activation of Gi /Go (A1) and Gi /Gq (A3).[4] In
humans, the A2A adenosine receptors have been expressed in a huge array of organs and tissues,
including heart, lung, liver, cardiovascular tissues, neutrophils, leukocytes, and endothelial cells
with remarkable implications in the regulation of inflammatory and immune responses.[5,6] On
account of these reasons, there is a growing interest in the A2A AR as a drug target.[5,7,8]
Agonists are explored as anti-inflammatory agents[5,9-12] and antagonists have emerged as an
attractive target to treat neurodegenerative diseases (Parkinson’s disease, Huntington’s, and
Alzheimer’s disease),[13-16] as well as in cancer immunotherapy.[3,8,17] A2A ARs are widely
expressed in several immune cell types: T cells, natural killer T (NLT) cells, dendritic cells
(DCs), monocytes and natural killer cells (NK).[3,17] In tumor microenvironment (TME)
augmentation in extracellular adenosine level enhances the signals of A2A AR present on immune
cells. Activation of A2A AR on T cells and NK cells causes immunosuppression by reducing their
proliferation, maturation, cytokine production (IL-2, IFN-γ) and tumor killing activity.[17-19]
Recent studies have highlighted/supported[7,20] that A2A AR knock out or use of A2A AR
antagonist in animal model has exhibited potent effects in increasing anti-tumor immunity and
suppresses tumor growth.[21-24]
3

Cl
O
N
N
N
N
N
N
N
N
N
N
N
Br
N
Me
F
H2N
N
N
H2N
H2N
N
N
O
NIR178
Ciforadenant
(CPI-444)
AZD-4635
Phase II
Phase I/II
Phase I/II
O
NH2
N
O
N
O
N
N
N
O
N
N
N
N
N
N
O
H2N
N
N
NH2
Vipadenant
Preladenant
(MK-3814)
Phase I
Preclinical
Fig. 1. A2A AR antagonist candidates in clinical trials for cancer immunotherapy.
Currently some A2A AR antagonists are in clinical trial for cancer immunotherapy either alone or
in combination with other immunotherapies (Fig 1).[25-27]
2. Design
Owing to our interest in adenosine receptors, we approached to design and synthesize novel A2A
receptor antagonists based on the structures reported in literatures. Recently Falsini M. et al and
Poli et al reported 1,2,4-triazolo[4,3-a] pyrazin-3-one (1 and 2) and imidazo[1,2-a]pyrazin-8-
amine (3) derivatives respectively as adenosine A2A R antagonists.[28-30] Similarly G. Yao and
H. Peng et al reported 1,2,4-triazolo[1,5-c]pyrimidin-5-amine derivatives (4 and 5).[31,32]
Taking the advantage of these bicyclic scaffolds, we designed novel structurally diversified
tricyclic benzo[4,5] imidazo[1,2-a] pyrazin-1-amine (6) core structure (Fig 2) to investigate the
antagonism of its derivatives in the adenosine A2A AR pathway.
4

Fig. 2. Design of benzo[4,5] imidazo[1,2-a]pyrazin-1-amine scaffold.
3. Results and Discussion
3.1. Chemistry
Synthesis of imidazo[1,2-a]pyrazine has been reported in literature.[33] However, to the best of
our knowledge no synthetic chemistry has been reported to construct benzo[4,5]imidazo[1,2-
a]pyrazin-1-amine. We designed our synthetic strategy from o-phenylenediamine (7) to achieve
the target products in four steps (Scheme 1). o-Phenylenediamine was treated with methyl 2,2,2-
trichloroacetimidate in AcOH to give intermediate 8 in 87 % yield[34] which was further treated
with ammonia solution to yield the corresponding 1H-benzo[d]imidazole-2-carbonitrile (9) in
83% yield.[35] Subsequently N-alkylation of 9 and 6 with the corresponding α-haloketones in
different solvents such as DMF, DCM and CH3CN, in the presence of variety of bases furnished
the precursor intermediate 1-(2-oxo-2-phenylethyl)-1H-benzo[d]imidazole-2-carbonitrile (10a-
10s). Recently we have optimized the cyclization condition for the formation of target
compounds and the methodology is ready to publish in another manuscript. As a matter of
interest we are here, showing only the optimized condition for this cyclization reaction.
Treatment of compound 10 with ammonium acetate in acetic acid resulted in the target
compounds (11-17, 19-30) in moderate to good yield (Table 1). Treatment of compound 17 with
BBr3 in DCM (Scheme 1) provided target compound 18 (Table 1).
5

Scheme 1. Reagents and conditions: a) methyl 2,2,2-trichloroacetimidate, AcOH, rt, 87%. b) 0 ±
o
0.4M) NH3 in dioxane and (7.0 M) NH3 in MeOH, sealed tube, 0 C to rt, 83%. c) RCOCH2X,
base, solvent. d) NH4OAc, AcOH, sealed tube 90 oC. e) BBr3, DCM, N2, 0 oC to rt, 50%.
Table 1. Synthesized compounds based on Benzo[4,5]imidazo[1,2-a]pyrazin-1-amine scaffold.
NH2
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
O
N
N
Br
N
O
N
N
N
N
14
13
12
15
11
NH2
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
20
17
19
O
HO
Cl
16
18
Br
NH2
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
F
N
N
N
N
N
N
N
N
21
F3CO
O2N
24
F
22
25
F3C
23
NH2
NC
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
O
N
F
N
N
N
O
O2N
N
28
F
26
27
30
29
6

Further, compounds 24 and 30 were modified to obtain N-acetylated compounds 37 and 38 for
the screening purpose (Scheme 2). First, protection of amine group as di-Boc (31/32) by treating
24 or 30 with Boc2O was performed. Nitro group was then reduced to amine with H2 in the
presence of Pd/C in methanol at room temperature, followed by acylation furnished the
intermediate (35/36). Di-Boc was removed with TFA to yield the target compounds 37 and 38 in
50% and 57% yield respectively.
o
Scheme 2. Reagents and conditions: a) Boc2O, TEA, DMAP, THF, 0 C to rt, 70% b) H2, 10%
Pd/C, MeOH, r.t. c) Ac2O, TEA, DMAP, DCM, 0 oC to rt, two steps 50% d) TFA, DCM 0 oC to
rt, 50-57%.
To study the effect of aliphatic substituents on the A2A AR binding affinity we synthesized
compounds 43, 44 and 45 (Scheme 3). The synthesis was commenced by α-bromination of tert-
butyl 4-acetylpiperidine-1-carboxylate (39) using Br2, offered intermediate 40 in 74% yield,
which was further used for N-alkylation on 1H-benzo[d]imidazole-2-carbonitrile to give
intermediate 41 in 66% yield. Cyclization of 41 in the presence of ammonium acetate furnished
compound 42 in 70% yield. Compound 42 was treated with TFA in DCM to obtain 43 in 60%
yield and alkylation was done using different alkyl halides to obtain compound 44 and 45 in 30%
and 43% yield respectively.
7

o
Scheme 3. Reagents and conditions: a) LHMDS, TMSCl, Br2, THF, -78 to 0 C, 74% b) 9,
K2CO3, DMF, 0 oC to rt, 66% c) NH4OAc, AcOH, sealed tube, 90 oC 70% d)TFA, DCM 0 oC to
rt, 60 % e) RX, K2CO3, DMF 0 oC to rt, 44 and 45 in 30% and 43%.
The synthesis of compound 50 (Scheme 4) started from 3-bromoprop-1-yne (46), which was
treated with (diacetoxyiodo)benzene in acetonitrile at room temperature to provide 47 in 45%
yield. Intermediate 47 was used for N-alkylation on 1H-benzo[d]imidazole-2-carbonitrile
afforded intermediate 48 in 70% yield. Cyclization of 48 with ammonium acetate in acetic acid
furnished compound 49 in 70% yield, which was further hydrolyzed to offer compound 50 in
60% yield.
Scheme 4. Reagents and conditions: a) PhI(OAc)2, AgOAc, H2O, ACN, rt, 45% b) 9, DIPEA, ACN, - 20
oC, 70% c) NH4OAc, AcOH, sealed tube, 90 oC 70% d) K2CO3, MeOH, 0 oC to rt 60%.
3.2. Biology studies
3.2.1. A2A AR binding assay
The biological activity of the synthesized benzo[4,5]imidazo[1,2-a]pyrazin-1-amine derivatives
toward hA2A receptor were first evaluated in an in-vitro receptor binding assay. Competitive
binding experiments were performed using membrane preparation of the human recombinant
A2A AR overexpressed from HEK-293 cells and [3H]-ZM241385 was used as a radioligand. The
binding data of synthesized compounds are listed in Table 2.
8

Sensible activity was observed for compound 11 comparable to its analogue compound 12 with
an ortho-OMe substituent. Encouraging results were obtained for meta-substituted -OMe, -Br
and -NHAc compounds (Table 2, compound 13, 14 and 38). In continuation para-position of the
ring was also analyzed with various electron donating and withdrawing groups. Although the
weakly electron donating groups such as methyl and tert-butyl groups were found to be
promising (Table 2, compound 15 and 16) yet same was not noticed in case of the moderate to
strong electron donating groups such as -NHAc, -OMe, and –OH (Table 2, compound 37, 17,
18). Halo substituted analogues chloro, bromo, 2,6 di-fluoro–CF3 and –OCF3 analogues
demonstrated moderate to inferior results (Table 2, compound 19, 20, 21, 22 and 23). Electron
withdrawing groups such as cyano and nitro showed lower activity compared with unsubstituted
analogue (Table 2, compound 24 and 25).
Table 2. Binding affinity data IC50 (nM) for compounds against human adenosine A2A receptorsa.
hA2A (nM)
hA2A (nM)
71.90 ± 0.90
74.40 ± 0.96
Comp
11
R
Comp
24
R
46.50 ± 0.98
O
83.10 ± 0.99
12
25
10.10 ± 0.96
10.10 ± 0.98
32.60 ± 0.97
13
14
15
16
26
27
37
38
9.20 ± 0.97
311.80 ± 0.83
48.60 ± 0.97
14.40 ± 0.99
33.00 ± 0.95
51.20 ± 0.96
>10000
17
43
9

81.30 ± 0.95
35.40 ± 0.98
18
19
>10000
44
45
50
>10000
149.00 ± 0.88
38.40 ± 0.97
33.70 ± 0.94
20
21
27.00 ± 0.99
113.70 ± 0.84
28
29
26.50 ± 0.92
130.80 ± 0.98
22
23
aData are expressed as means ± SEM.
The aryl heterocyclic furan analogue (Table 2, compound 26) displayed comparable A2AR
binding as phenyl compound. However, heterocyclic piperidine and N-substituted piperidine at
3-position revealed slumped activity (Table 2, compound 43, 44 and 45). Replacement of the
phenyl ring at 3-position by CH2OH group further reduced the activity (Table 2 compound 50). It
has been noticed that the activity was further suppressed by introducing methyl group at 4-
position of benzo[4,5]imidazo [1,2-a]pyrazin-1-amine (Table 2 compound 29). Interestingly it
was observed that when aryl was replaced with benzyl (on carbon atom spacer) moiety, 5-fold
increased A2AR binding affinity (Table 2, compound 27) was observed compared with phenyl
analogue (Table 2, compound 11).
3.2.2. A1 AR binding assay
While all of the synthesized compounds were screened for A2A binding affinity, a few potent A2A
binders were selected for the selectivity evaluation against human A1 adenosine receptor. In-vitro
competitive binding experiments with the hA1 CHO-K1 membranes were performed using [3H]
DPCPX as a radioligand. Among all the compounds screened, p-tolyl (15), 3-bromophenyl (14),
3-methoxyphenyl (13) and 7,8-difluoro-3-(3-methoxyphenyl) (28) showed low selectivity of A2A
AR to A1 AR, however the A2A binding affinity of benzyl substituted analogue (27) was 80-fold
more potent than A1. Interestingly, the analogues with furan-2-yl (26), 3-acetamidophenyl (38)
and 2,4-difluorophenyl (21) displayed higher binding affinity to A1 AR than to A2A AR.
10

Table 3. Binding affinity data for selected compounds against human adenosine A2A and A1 receptorsa.
hA2A IC50 (nM)
A1 IC50 (nM)
Compound
A1/A2A
9.5
10.10 ± 0.96
10.10 ± 0.98
14.40 ± 0.99
33.70 ± 0.94
32.30 ± 0.97
9.20 ± 0.97
27.00 ± 0.99
48.60 ± 0.97
95.80 ± 0.96
92.10 ± 0.98
63.90 ± 0.92
4.40 ± 0.93
13
9.1
14
4.4
15
0.13
0.20
79.7
17.0
0.35
21
6.30 ± 0.98
26
733.40 ± 0.97
460.30 ± 0.99
17.00 ± 0.87
27
28
38
aData are expressed as means ± SEM.
3.2.3. cAMP functional assay
Over-activation of A2A AR signal in tumor microenviroment triggers the accumulation of
intracellular cAMP through stimulation of intracellular adenylyl cyclase. The rise in intracellular
cAMP acting primarily through protein kinase A (PKA) has a broad range of
immunosuppressive effects.[36] Therefore, the ability to inhibit or stimulate hA2A AR could be
determined by evaluating their effect on cAMP production in human HEK-293 cells which
stably express hA2A AR.
Table 4. IC50 of selected compounds on cAMP assays in human HEK-293 cellsa.
hA2A IC50 (nM)
10.10 ± 0.98
9.20 ± 0.97
cAMP IC50 (nM)
Compound
83.00 ± 0.95
14
27
28
31.00 ± 0.86
77.00 ± 0.84
27.00 ± 0.99
aData are expressed as means ± SEM.
Fig. 3. The IC50 curve of compound 27 in cAMP functional assay.
11

We selected compound 14, 27 and 28, which show potent hA2A AR binding affinity and
selectivity, to assess their antagonistic activities in A2A AR mediated cAMP functional assay. See
Table 4 for the cyclic AMP assay results. The functional assay data for the tested compounds
reflected A2A AR antagonist potency. Expectedly, compound 27 with highest hA2A AR
antagonistic affinity (IC50 9 nM of A2A AR binding affinity) showed most potent functional assay
data with the IC50 value 31 nM (Fig 3, Table 4). Compound 28 displayed less inhibition of
cAMP production compared to compound 27, consistent with its lower binding affinity to the
receptor. However, compound 14 is more than 2-fold less active than compound 27 in inhibiting
cAMP production despite both of these two compounds display comparable potency of A2A AR
binding affinity.
3.2.4. T Cell activation assay
The previous studies strongly suggested that activation of A2A AR on T-cell decrease the
cytokines (IL-2 and IFN-γ) production and suppress anti-tumor immune response.[17-19] In this
study we examined the efficiency of A2A AR antagonist compounds on T-cell activation (IL-2
production). Three compounds (14, 27 and 28) were evaluated in this assay and the results are
listed in Table 5. Compound 14 and 28 showed sensible activity of the IL-2 production with
EC50 376 nM and 555 nM respectively. It is worth noting that compound 27 shows admirable
activity with EC50 165 nM (Fig 4). Th1 cytokines such as IL-2 and IFN-γ stimulate the
differentiation and activation of cytotoxic lymphocytes that are responsible for the cell-mediated
immune responses against viruses and tumor cells. IL-2 is expressed by activated T cells, but not
resting T cells, making it a surrogate for T-cell activation. Our compounds are able to stimulate
IL-2 production by T cell activation, suggesting its potential as an immunotherapy to activate T
cells for tumor killing.
Table 5. EC50 of selected compounds of IL-2 production on T-Cell activationa.
hA2A IC50 (nM)
cAMP IC50 (nM)
EC50 (nM)
Compound
10.10 ± 0.98
9.20 ± 0.97
27.00 ± 0.99
83.00 ± 0.95
31.00 ± 0.86
77.00 ± 0.84
14
27
28
376.40 ± 0.95
164.60 ± 0.34
523.60 ± 0.66
aData are expressed as means ± SEM.
12

Fig. 4: The EC50 curve of the IL-2 production of compound 27.
3.2.5. Molecular docking studies
We performed the molecular docking modeling to interpret the hA2A AR selectivity to the
synthesized compound 27 at the molecular level. The binding modes of compound 27 at the
hA2A AR cavity were analyzed by docking simulations using the Schrodinger software (LLC,
New York, NY, 2017). The docking analysis was carried out by induced fit docking module[37]
associated with Glide XP algorithm[38] in Schrodinger package, and the crystal structure of
hA2A AR in complex with antagonist ZM241385 (PDB ID: 5NM4)[39] was used. In addition, the
binding mode of hA1 AR was also analyzed with the same docking protocols by Schrodinger.
The docking results (Fig. 5) reveal that compound 27 adopts the general binding mode[28,29,39]
at both hA2A AR and hA1 AR binding sites. In this binding mode, the benzo[4,5]imidazo[1,2-
a]pyrazin-1-amine scaffold positions in the depth of the binding pocket and gives π-π interaction
with the Phe residue (Phe177 in hA2A AR, Phe278 in hA1 AR) (Fig. 5). In addition, the scaffold
forms H-bond with the Asparagine residue in both hA2A AR (Asn358) and hA1 AR (Asn361)
(Fig. 5), and it forms an additional H-bond with Glu178 in hA2A AR (Fig. 5a, 5b). The
substituted benzyl group is located in proximity of Leu176, Ile175, Ser76, Tyr119, Leu372,
Ile379 and Met375 at the entrance of hA2A AR cavity (Fig. 5a, 5b). The nonpolar profile of these
cited residues at hA2A AR cavity allows favorable interactions with the hydrophobic benzyl
group. Whereas, the more polar profile of the corresponding hA1 AR residues (Glu277, Ile176,
Asn177, Tyr119, Ile381, His385 and Thr377) allows unfavorable interaction with the
hydrophobic benzyl group (Fig. 5, c, d). Therefore, the compound 27 adopts a much more
favorable binding pose at hA2A AR cavity than at hA1 AR cavity. In addition, the Glide XP
docking results also indicate that the binding pose is associated with a better docking score at
hA2A AR (-14.14 kcal/mol) than at hA1 AR (-11.46 kcal/mol). Hence, the molecular docking
results explain the hA2A AR affinity and selectivity of the compound 27.
13

Fig. 5. Schematic description of the ligand-target interaction between compound 27 and hA2A
AR (a), hA1 AR (b) (built with MOE software).[40] The binding mode of compound 27 at the
hA2A AR (c), hA1 AR (d) binding cavity with indication of some key receptor residues.
4. Conclusion
In present study, we designed and synthesized a novel benzo[4,5]imidazo [1,2-a]pyrazin-1-
amine scaffold from which a serial of derivatives were prepared and screened in sets of
biological assays relevant to A2A AR pathway using ZM 241385 as a benchmark reference (A2A
AR IC50 3.9 nM, cAMP IC50 5 nM, T cell activation EC50, 72.3 nM), a potent and selective
adenosine A2A R antagonist which was widely used as a tool compound in the study of A2A AR
vivo biology. In-vitro evaluations of the A2A AR, A1 AR, cAMP and T cell assays for the
synthesized compounds showed promising results. The compound 27 showed potent binding
affinity to A2A AR (IC50 9.2 nM), good selectivity against A1 AR (A2A/A1 80 fold) and high
potency in cAMP (IC50 31.0 nM) functional and IL-2 (EC50 164.6 nM) production assays. With
these encouraging results we anticipate that this novel benzo[4,5] imidazo[1,2-a]pyrazin-1-amine
scaffold could be an excellent starting point for further development of A2A AR antagonists to
14

benefit the field of cancer immunotherapy. Current effort is focused on further improving
potency and selectivity against A2A AR and the findings will be reported in due course.
5. Experimental section
5.1. Chemistry
All reactions were performed under mentioned conditions. Analytical thin layer chromatography
was performed using TLC pre-coated silica gel 60 F254 (20 x 20 cm). TLC plates were visualized
by exposing UV light or by iodine vapors or immersion in anisaldehyde charring reagent or in
2,4-dinitrophenyl hydrazine or ninhydrin followed by heating on hot plate. Organic solvent were
concentrated by rotary evaporation and dried using high vacuum suction pump. Compounds were
1
purified by column chromatography (flash silica gel chromatography). H NMR spectra were
recorded with 400 and 500 MHz NMR instruments. Chemical data for protons are reported in
parts per million (ppm, scale) downfield from tetramethylsilane and are referenced to the residual
proton in the NMR solvent (CDCl3: δ 7.26, DMSO-d6 δ 2.50 or other solvents as mentioned).
5.1.1. General procedure for the synthesis of compounds 8-9
5.1.1.1. 2-(trichloromethyl)-1H-benzo[d]imidazole (8)
In RBF o-phenylenediamine (10.0 g, 92.6 mmol) was dissolved in 120 mL of AcOH. This
o
solution was cooled to 5-10 C and to this solution was added methyl 2,2,2-trichloroacetimidate
drop-wise(12.48 mL, 97.23 mmol) (Note: Reaction is exothermic, so initially a few mL of
methyl 2,2,2-trichloroacetimidate was added very slowly and white precipitate formed during
addition). After addition the reaction was allowed to stir at room temperature for 1.5h and the
reaction was monitored by TLC. After completion of the reaction, the mixture diluted with 25
mL of water and the resulting mixture was filtered. The solid cake was washed with water
1
(3x25mL) and dried on high vacuum to give white product (19.0 g, 87%). H NMR (DMSO-d6,
500 MHz, δ ppm): 7.67 (2H, s), 7.34 (2H, dd, J=6.0, 3.1Hz); LCMS (M+1)+, calculated: 236.49;
found: 236.89.
5.1.1.2. 1H-benzo[d]imidazole-2-carbonitrile (9)
In a sealed tube 2-(trichloromethyl)-1H-benzo[d]imidazole (5.0 g, 21.3 mmol) was taken flushed
with nitrogen and cooled to 0 oC. 0.4 M NH3 in dioxane (107 mL, 42.7 mmol) was added under
15

o
nitrogen slowly at 0 C. After addition the sealed tube was closed tightly and the reaction
mixture allowed stirring at room temperature for 1h. After stirring for 1h the reaction mixture
o
was cooled to 0 C and 7.0 M NH3 in MeOH (6.1 mL, 42.7 mmol) was then added under
nitrogen. After addition the tube was sealed tightly and the reaction mixture was allowed stirring
at room temperature for 2h and reaction progress was monitored by TLC. After completion of
reaction, the reaction mixture was filtered and the solid collected was then dissolved in EtOAc
and water. Organic layer was separated and the aqueous layer was re-extracted with EtOAc
(3x50 mL). The combined organic extracts was dried over Na2SO4 and concentrated. The filtrate
was concentrated under reduced pressure. The resulted solid was washed with solvent mixture of
ether/hexane (3/1) and ether. The solid was collected and dried on high vacuum. Yield: (2.5 g,
1
83 %). H NMR (400 MHz, MeOH-d4, δ ppm): 7.6 (2H, m), 7.4 (2H, m), LCMS (M+
H
)+，
calculated: 144.15; found 144.11.
5.1.2. General procedure for the synthesis of compounds 10a-10s
In an oven dried RBF 1H-benzo[d]imidazole-2-carbonitrile (50 mg, 0.34 mmol) was taken in dry
DMF (1.5 mL). This mixture was cooled to 0 oC and K2CO3 (96 mg, 0.69 mmol) was added. The
reaction mixture was stirred for 5 minutes and to this reaction 2-bromo-1-phenylethan-1-one (76
mg, 0.38 mmol) was then added. The reaction mixture was allowed stirring at room temperature
and reaction progress was monitored by TLC. After completion of reaction, the reaction mixture
was diluted with EtOAc/water. Organic layer was separated and aqueous layer was re-extracted
with EtOAc (3x20 mL). The combined organic extracts was washed with brine, dried over
Na2SO4 and concentrated. The resulted residue was chromatographed (flash silica gel
chromatography) to give the desired product of 1-(2-oxo-2-phenylethyl)-1H-benzo[d]imidazole-
2-carbonitrile (10a) with 70% yield. The synthesis, NMR data and structures of N-alkylation
intermediates (10a-10s) is available in the supporting information section.
5.1.3. General procedure for the synthesis of compounds 11-29, 37-38, 43-45, 50
In a sealed tube NH4OAc(36 mg, 0.45 mmol) was added a solution of 1-(2-oxo-2-phenylethyl)-
1H-benzo[d] imidazole-2-carbonitrile (40 mg, 0.15 mmol) in AcOH (1.0 mL). After addition the
o
tube was sealed tightly and reaction mixture was allowed stirring at 90 C and reaction progress
was monitored by TLC (reaction time contrast for different substitutions). After completion of
16

reaction, the reaction mixture was cooled to room temperature and neutralized with saturated
NaHCO3 solution and extracted with EtOAc (20 mL). Organic layer was separated and aqueous
layer was re-extracted with EtOAc (2x20 mL). The combined organic extracts was washed with
brine, dried over Na2SO4 and concentrated. The resulted residue was chromatographed (flash
silica gel chromatography).
5.1.3.1. 3-Phenylbenzo[4,5]imidazo[1,2-a]pyrazin-1-amine (11)
1
Yield: (29.8 mg, 75%). H NMR (400 MHz, DMSO-d6, δ ppm): 8.93 (1H, s), 8.38 (1H, d, J =
13
8.1 Hz), 8.10 (2H, d, J = 7.7 Hz), 7.91 (1H, d, J = 8.1 Hz), 7.49 (7H, m). C NMR (101 MHz,
DMSO, δ ppm): 150.20, 143.56, 137.66, 135.75, 135.48, 130.36, 128.94, 128.24, 126.08, 125.98,
122.78, 120.60, 113.30, 106.32. LCMS (M+1)+, calculated: 261.30, found: 261.37.
5.1.3.2. 3-(2-Methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (12)
1
Yield: (30.3 mg, 76%). H-NMR (500 MHz, CDCl3, δ ppm): 8.49 (1H, s), 8.08 – 7.93 (2H, m),
7.87 (1H, d, J = 8.2 Hz), 7.54 (1H, t, J = 7.5 Hz), 7.37 (2H, ddd, J = 15.6, 11.8, 4.6 Hz), 7.15 –
6.99 (2H, m), 6.05 (2H ,s), 3.95 (3H, s). 13C NMR (101 MHz, CDCl3) δ 156.74, 149.08, 143.70,
135.10, 133.06, 130.55, 129.81, 129.23, 125.92, 125.76, 122.60, 121.11, 120.80, 111.48, 111.38,
110.17, 55.74. LCMS (M+1)+, calculated: 291.33, found: 291.71
5.1.3.3. 3-(3-Methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (13)
1
Yield: (29.8 mg, 75%). H-NMR (500 MHz, DMSO-d6, δ ppm): 8.93 (1H, s), 8.38 (1H, d, J =
8.1 Hz), 7.90 (1H, d, J = 8.2 Hz), 7.67 (2H, dd, J = 8.9, 5.0 Hz), 7.54 (1H, t, J = 7.6 Hz), 7.47
(1H, t, J = 7.6 Hz), 7.39 (3H, dd, J = 9.5, 6.2 Hz), 6.94 (1H, dd, J = 8.1, 2.4 Hz), 3.85 (3H, s).
13C NMR (126 MHz, DMSO) δ 160.08, 150.09, 143.56, 139.14, 135.52, 130.36, 129.96, 126.00,
122.77, 120.60, 118.46, 113.70, 113.33, 111.71, 106.57, 55.64.LCMS (M+1)+,calculated: 291.33,
found: 291.64.
5.1.3.4. 3-(3-Bromophenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (14)
1
Yield: (27.9 mg, 70%). H NMR (400 MHz, CDCl3, δ ppm): 8.18 (1H, s), 8.13 (1H, t, J = 1.8
Hz), 8.02 – 7.83 (3H, m), 7.62 – 7.44 (3H, m), 7.35 (1H, t, J = 7.9 Hz), 5.85 (2H, s). 13C NMR
(126 MHz, DMSO, δ ppm): 150.20, 143.56, 140.08, 135.51, 133.98, 131.09, 130.79, 130.41,
17

128.58, 126.11, 124.76, 122.98, 122.66, 120.65, 113.30, 107.11. LCMS (M+1)+, calculated:
339.19, found: 339.19, 341.13
5.1.3.5. 3-(p-Tolyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (15)
1
Yield: (30.6 mg, 77%). H-NMR (400 MHz, CDCl3, δ ppm): 8.14 (1H, s), 8.03 – 7.78 (4H, m),
7.59 – 7.52 (1H, m), 7.45 (1H, t, J = 7.3 Hz), 7.29 (2H, d, J = 8.0 Hz), 5.90 (2H, s), 2.42 (3H, s).
13C NMR (126 MHz, CDCl3) δ 149.30, 143.66, 138.22, 137.01, 135.13, 134.30, 129.79, 129.54,
126.03, 126.01, 122.82, 120.95, 111.36, 105.49, 21.27. LCMS (M+1)+, calculated: 275.32, found:
275.23
5.1.3.6. 3-(4-(tert-Butyl)phenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (16)
1
Yield: (28.7 mg, 72%). H-NMR (400 MHz, CDCl3, δ ppm): 8.17 (1H, s), 8.04 – 7.80 (4H, m),
13
7.64 – 7.43 (4H, m), 5.92 (2H, s), 1.40 (9H, s). C NMR (126 MHz, CDCl3) δ 151.47, 149.37,
143.63, 137.06, 135.14, 134.34, 129.77, 126.03, 125.91, 125.79, 122.81, 120.92, 111.36, 105.54,
34.69, 31.35. LCMS (M+1)+, calculated: 317.40, found: 317.30
5.1.3.7. 3-(4-Methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (17)
1
Yield: (31.0 mg, 78%). H-NMR (400 MHz, CDCl3, δ ppm): 8.10 (1H, s), 7.97 (1H, d, J = 8.3
Hz), 7.94 – 7.83 (3H, m), 7.59 – 7.53 (1H, m), 7.49 – 7.43 (1H, m), 7.05 – 6.97 (2H, m), 5.86
(2H, s), 3.88 (3H, s). 13C NMR (101 MHz, CDCl3) δ 159.88, 149.31, 143.52, 136.84, 134.98,
129.70, 127.41, 126.03, 122.81, 120.87, 114.22, 111.37, 104.91, 55.40. LCMS (M+1)+,
calculated: 291.32, found: 291.74
5.1.3.8. 4-(1-Aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)phenol (18)
3-(4-Methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (17) 0 ± 0.20g, 0.67 mmol)) was
taken up in DCM. The reaction vessel was then transferred to an ice bath and BBr3 0 ± 0.13 mL,
38 mmol) is added slowly dropwise. After the addition is complete, the reaction mixture is
allowed to stir at room temperature for 8h. After completion of the reaction, as indicated by TLC,
the reaction vessel is kept un-corked, whereby the excess BBr3 is neutralized by atmospheric
moisture. It was then diluted with water and extracted with ethyl acetate. The ethyl acetate
extracts are combined, dried over anhydrous Na2SO4, filtered and concentrated in vacuo and
18

column chromatographed to get the target compound, 4-(1-Aminobenzo[4,5]imidazo[1,2-
a]pyrazin-3-yl)phenol (18) 0 ± 0.094 g, 50%). 1H NMR (400 MHz, DMSO-d6, δ ppm): 8.46 (1H,
s), 8.03 (1H,s), 7.83 (2H, dd, J = 18.1, 8.1 Hz), 7.50 (1H, t, J = 7.7 Hz), 7.41 (1H, t, J = 8.1 Hz),
5.93 (2H, s). LCMS (M+1)+, calculated: 277.29, found: 277.47.
5.1.3.9. 3-(4-Chlorophenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (19)
1
Yield: (27.9 mg, 70%). H-NMR (500 MHz, DMSO-d6, δ ppm): 9.00 (1H, s), 8.38 (1H, d, J =
8.1 Hz), 8.14 (2H, d, J = 8.1 Hz), 7.93 (1H, d, J = 8.1 Hz), 7.63 – 7.42 (6H, m). 13C NMR (126
MHz, DMSO) δ 150.21, 143.57, 136.57, 135.45, 134.51, 132.77, 130.37, 128.92, 127.68, 126.02,
122.88, 120.63, 113.25, 106.64. LCMS (M+1)+, calculated: 295.74, found: 295.16
5.1.3.10. 3-(4-Bromophenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (20)
1
Yield: (29.5 mg, 70%). H-NMR (400 MHz, DMSO-d6, δ ppm): 9.00 (1H, s), 8.36 (1H, d, J =
8.1 Hz), 8.06 (2H, d, J = 8.6 Hz), 7.91 (1H, d, J = 8.1 Hz), 7.69 (2H, d, J = 8.6 Hz), 7.58 – 7.37
(4H, m). 13C NMR (101 MHz, CDCl3) δ 149.33, 143.63, 136.04, 135.80, 135.03, 131.91, 129.77,
127.63, 126.23, 123.11, 122.42, 121.04, 111.32, 105.91. LCMS (M+1)+, calculated: 339.19,
found: 339.20, 341.18.
5.1.3.11. 3-(2,4-Difluorophenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (21)
Yield: (25.9 mg, 65%). 1H NMR (400 MHz, CDCl3, δ ppm): 8.30 (1H, s), 8.09 (1H, dd, J = 15.8,
8.8 Hz), 7.88 (2H, dd, J = 25.3, 8.3 Hz), 7.45 (2H, m), 6.98 – 6.84 (2H, m), 5.76 (2H, s). LCMS
(M+1)+, calculated: 297.28, found: 297.21.
5.1.3.12. 3-(4-(Trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2-I]pyrazin-1-amine (22)
1
Yield: (27.9 mg, 70%). H NMR (400 MHz, CDCl3, δ ppm): 8.27 (1H, s), 8.09 (2H, d, J = 8.1
Hz), 8.02 (1H, d, J = 8.2 Hz), 7.95 (1H, d, J = 8.1 Hz), 7.75 (2H, d, J = 8.3 Hz), 7.61 (1H, t, J =
7.7 Hz), 7.51 (1H, t, J = 7.6 Hz), 5.89 (2H, s). LCMS (M+1)+, calculated: 329.29, found: 329.74
5.1.3.13. 3-(4-(Trifluoromethoxy)phenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (23)
19

1
Yield: (29.9 mg, 75%). H NMR (400 MHz, CDCl3, δ ppm): 8.15 (1H, s), 8.10 – 7.79 (4H, m),
7.58 (1H, t, J = 7.7 Hz), 7.48 (1H, t, J = 7.7 Hz), 7.34 (2H, d, J = 8.3 Hz), 6.04 (2H, s). LCMS
(M+1)+, calculated: 345.29, found: 345.25.
5.1.3.14. 3-(4-nitrophenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (24)
Yield: (21.5 mg, 79%). 1H NMR (400 MHz, DMSO-d6, δ ppm): 9.21 (1 H, s), 8.41 – 8.34 (5 H,
m), 7.93 (1 H, d, J= 8.1Hz), 7.59 – 7.48 (4 H, m). HRMS [M+H]+, C16H11N5O2, calculated:
306.0986, found: 306.0986
5.1.3.15. 4-(1-Aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)benzonitrile (25)
1
Yield: (21.6 mg, 55%). H NMR (400 MHz, CDCl3, δ ppm): 8.29 (1H, s), 8.18 – 7.90 (4H, m),
7.78 (2H, d, J = 8.2 Hz), 7.61 (1H, t, J = 7.7 Hz), 7.55 – 7.50 (1H, m), 5.89 (2H, s). LCMS
(M+1)+, calculated: 286.31, found: 286.22
5.1.3.16. 3-(Furan-2-yl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (26)
1
Yield: (27.8 mg, 70%). H NMR (400 MHz, DMSO-d6, δ ppm): 8.63 (1H, s), 8.37 (1H, d, J =
8.1 Hz), 7.90 (1H, d, J = 8.2 Hz), 7.78 (1H d, J = 0.9 Hz), 7.65 – 7.28 (4H m), 6.83 (1H, d, J =
3.0 Hz), 6.63 (1H, dd, J = 3.3, 1.8 Hz). 13C NMR (126 MHz, DMSO-d6, δ ppm): 152.65, 150.56,
143.39, 143.11, 135.40, 130.33, 129.33, 126.00, 123.02, 120.59, 113.34, 112.42, 107.46, 104.75.
LCMS (M+ H)+, calculated: 251.26, found: 251.55.
5.1.3.17. 3-Benzylbenzo[4,5]imidazo[1,2-a]pyrazin-1-amine (27)
Yield: (27.4 mg, 69%). 1H NMR (400 MHz, CDCl3, δ ppm): 7.94 (1H. d, J = 8.3 Hz), 7.75 (1H,
d, J = 8.3 Hz), 7.52 (2H, dd, J = 11.7, 4.5 Hz), 7.44 – 7.25 (6H, m), 5.83 (2H, s), 4.01 (2H, s).
13C NMR (101 MHz, CDCl3, δ ppm): 149.64, 143.41, 138.72, 138.35, 134.92, 129.49, 129.19,
128.67, 126.69, 125.81, 122.70, 120.80, 111.32, 107.18, 41.13. LCMS (M+1)+, calculated:
275.32, found: 275.66
5.1.3.18. 7,8-Difluoro-3-(3-methoxyphenyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (28)
Yield: (21.9 mg, 55%). 1H NMR (400 MHz, CDCl3, δ ppm): 8.03 (1H, s), 7.71 (2H, m), 7.48 (2H,
dd, J = 9.7, 5.0 Hz), 7.39 (1H, t, J = 7.9 Hz), 6.95 (1H, dd, J = 8.1, 2.5 Hz), 5.84 (2H s), 3.91 (3H
20

13
s). C NMR (126 MHz, CDCl3) δ 160.14, 148.92, 138.21, 137.41, 129.87, 118.36, 114.21,
111.88, 107.96, 107.80, 105.66, 99.29, 99.11, 55.42. LCMS (M+1)+, calculated: 327.30, found:
327.26
5.1.3.19. 4-Methyl-3-phenylbenzo[4,5]imidazo[1,2-a]pyrazin-1-amine (29)
Yield: (25.9 mg, 65%). 1H NMR (500 MHz, CDCl3, δ ppm): 8.12 (1H, d, J = 8.5 Hz), 7.94 (1H,
d, J = 8.3 Hz), 7.52 – 7.44 (3H, m), 7.41 (2H, t, J = 7.5 Hz), 7.33 (2H, t, J = 7.6 Hz), 5.72 (2H, s),
2.86 (3H s). 13C NMR (101 MHz, CDCl3, δ ppm): 147.86, 143.98, 138.71, 136.04, 135.48,
131.39, 129.86, 128.37, 127.87, 125.38, 122.62, 120.88, 119.50, 115.10, 17.23. LCMS (M+1)+,
calculated: 275.32, found: 275.45.
5.1.3.20. N-(4-(1-aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)phenyl)acetamide (37)
1H NMR (400 MHz, DMSO-d6, δ ppm): 10.05 (1 H, s), 8.85 (1 H, s), 8.35 (1 H, d, J =8.1Hz),
8.01 (2 H, d, J= 8.7Hz), 7.90 (1 H, d, J =8.1Hz), 7.69 (2 H, d, J =8.7Hz), 7.53 (1 H, t, J =7.6Hz),
7.46 (1 H, t, J= 7.6Hz), 7.36 (2 H, s), 2.08 (3 H, s). HRMS [M+H]+, C18H15N5O, calculated:
318.1349, found: 318.1346
5.1.3.21. N-(3-(1-aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)phenyl)acetamide (38)
1H NMR (500 MHz, DMSO-d6, δ ppm): 10.07 (1 H, s), 8.82 (1 H, s), 8.37 (1 H, d, J=8.2 Hz),
8.18 (1 H, s), 7.91 (1 H, d, J= 8.2 Hz), 7.71 (1 H, d, J= 7.8 Hz), 7.62 (1 H, d, J =7.9 Hz), 7.54 (1
H, t, J= 7.6 Hz), 7.47 (1 H, t, J =7.6 Hz), 7.42 – 7.33 (3 H, m), 2.08 (3 H, s). LCMS (M+Na)+,
calculated: 340.34, found: 340.50
5.1.3.22. 3-(piperidin-4-yl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (43)
1H NMR (500 MHz, MeOD, δ ppm): 8.07 (1 H, d, J =8.3 Hz), 7.95 (1 H, s), 7.86 (1 H, d, J= 8.3
Hz), 7.54 (1 H, t, J =7.7 Hz), 7.45 (1 H, t, J =7.7 Hz), 3.26 (2 H, d, J =12.5 Hz), 2.84 (2 H, t, J
=12.4 Hz), 2.78 – 2.70 (1 H, m), 2.04 (2 H, d, J =13.0 Hz), 1.89 – 1.78 (2 H, m). HRMS [M+H]+,
C15H17N5, calculated: 268.1557, found: 268.1559
5.1.3.23. 3-(1-(2-methoxyethyl)piperidin-4-yl)benzo[4,5]imidazo[1,2-a]pyrazin-1-amine (44)
21

1H NMR (400 MHz, MeOD, δ ppm): 8.05 (1 H, d, J= 8.3), 7.92 (1 H, s), 7.84 (1 H, d, J= 8.3
Hz), 7.54 – 7.49 (1 H, m), 7.45 – 7.40 (1 H, m), 3.60 (2 H, t, J =5.6 Hz), 3.37 (3 H, s), 3.21 (2 H,
d, J= 12.0 Hz), 2.75 (2 H, t, J =5.6 Hz), 2.65 – 2.54 (1 H, m), 2.35 (2 H, td, J= 12.0, 2.2 Hz),
2.06 – 1.99 (2 H, m), 1.98 – 1.86 (2 H, m). HRMS [M+H]+, C18H23N5O, calculated: 326.1975,
found: 326.1972
5.1.3.24.
2-(4-(1-aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)piperidin-1-yl)-1-
morpholinoethan-1-one (45)
1H NMR (500 MHz, CDCl3, δ ppm): 7.95 (1 H, d, J=8.1 Hz), 7.83 (1 H, d, J =8.2 Hz), 7.60 (1 H,
s), 7.53 (1 H, t, J= 7.5 Hz), 7.43 (1 H, t, J =7.4 Hz), 5.73 (2 H, s), 3.76 – 3.60 (7 H, m), 3.26 (2
H, s), 3.05 (2 H, d, J =10.3 Hz), 2.56 (1 H, t, J =11.6 Hz), 2.34 – 2.19 (2 H, m), 2.04 (2 H, d, J=
12.3 Hz), 1.91 – 1.79 (2 H, m). HRMS [M+H]+, C21H26N6O2, calculated: 395.2190, found:
395.2188.
5.1.3.25. (1-aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)methanol (50)
1H NMR (400 MHz, DMSO) δ 8.24 (1H, d, J = 8.2 Hz), 8.14 (1H, s), 7.87 (1H, d, J = 8.2 Hz),
7.54 – 7.47 (1H, m), 7.45 – 7.39 (1H, m), 7.23 (2H, s), 5.32 (1H, s), 4.45 (2H, s). HRMS [M+H]+,
C11H10N4O, calculated: 215.0927, found: 215.0929.
5.2. Biological activity
5.2.1. A2A AR binding assay
The synthesized compounds were tested to evaluate their affinity for the A2A AR expressed on
HEK293 cell membranes. The assay buffer (50 mM Tris-HCl of pH 7.4; 10 mM MgCl2; 1 mM
EDTA; 1 μg/mL adenosine deaminase) was prepared and stored at 4˚C for use. The wash buffer
(50 mM Tris-HCl of pH 7.4; 154 mM NaCl) was prepared and stored at 4˚C for use. 0.5% PEI
solution was prepared by dissolving 0.5 g of PEI in 100 mL of ddH2O and stored at 4˚C for use.
1 μL Of A2A membrane (hA2A HEK-293 membrane) and 300 μL of assay buffer was added into
96-well plate in which each well contains testing compound (10 different concentrations from
1000 nM to 0.05 nM by three-fold sequential dilution) and the plate was shaken for 5 minutes.
[3H]-ZM241385 (final concentration 0.5 nM) was then added to the assay well and the plate was
incubated for 1.5h at 27 oC. Bound radioactivity and free radioactivity were separated by filtering
22

the assay mixture through a UNIFILTER-96 GF/B filter plate (pre-incubated with 0.5% PEI for 1
h) and washed with wash buffer. ULTIMA GOLD (40 μL) was added to each well of the plate
and the count per minute (CPM) was read on TopCount.
5.2.2. A1 AR binding assay
The synthesized compounds were tested to evaluate their affinity for the A1 AR expressed on
CHO-K1 cell membranes. The assay buffer (50 mM Tris-HCl of pH 7.4; 10 mM MgCl2; 1 mM
EDTA; 1 μg/mL adenosine deaminase) was prepared and stored at 4˚C for use. The wash buffer
was prepared and stored at 4˚C for use. 0.5% PEI solution was prepared by dissolving 0.5 g of
PEI in 100 mL of ddH2O and stored at 4˚C for use. The CHO-K1 cells with adenosine A1
(human) membrane were purchased from Perkinelmer. The dilution of A1 membrane (hA1 CHO-
K1 membrane) was performed by dissolving 20 U of A1 membrane and 0.41 uCi [3H] DPCPX
(final concentration 2.5 nM) in 1 mL of assay buffer. 50 µL of diluted A1 membrane was then
added into 96-well plate in which each well contains testing compound (10 different
concentrations from 1000 nM to 0.05 nM by three-fold sequential dilution) and the plate was
shaken for 5 minutes. The plate was then incubated at 25 ˚C for 50 minutes. The membrane
mixture was filtered through UNIFILTER-96 GF/B filter plate (pre-incubated with 0.5% of PEI
at 4˚C for 1.5 hours and then washed twice with 1 mL of wash buffer per well) and washed four
times (4x500 µL for each well) with wash buffer. After the plate was dried at 55˚C for 10
minutes, 40 µL of ULTIMA GOLD was added to each well and the CPM was recorded by
TopCount.
5.2.3. Cyclic AMP functional assay
HEK293-A2A cells which express human A2A AR were cultured in a growth medium [this
medium contains DMEM (Gibco), 10% FBS (Gibco), 1X Penicillin-Streptomycin (Gibco), and
400μg/mL of G418 (Invitrogen)] at 37 °C under 5% CO2. The assay buffer in which contains
1xHBSS, 0.1% BSA (Perkin Elmer), 20mM HEPES (Gibco) and 100nM IBMX (Sigma) was
prepared and stored at room temperature for assay use. The 8x test compound stock solution and
8x NECA stock solution (12 nM) were prepared with assay buffer for use. 20X cAMP-d2 and
20X anti-cAMP-Eu3+ detection reagent solutions were prepared using lysis buffer. Briefly,
HEK293-A2A cells were seeded in 384-well plate (6007680-50, PE) in which each well contains
23

15,000 cells suspended in 15 µL of assay buffer. 2.5μL of test compound solution was added to
indicated well of the 384-well plate prepared above and incubated at 37 °C for 10 minutes. Next,
2.5μL of NECA stock solution was added to the 384-well plate and incubated at 37 °C for
additional 30 minutes (the final volume of the reaction system is 20μL). Finally, 10μL of cAMP-
d2 and 10μL of anti-cAMP-Eu3+ detection reagent were added into each well of the plate and
incubated at room temperature for 1h. The data was collected at the wavelength of 665 nm and
615 nm on Envision 2104 plate reader.
5.2.4. T-cell activation assay
T cells were isolated using Human Pan T Cell Isolation Kit (MicroBead No. 130-096-535) from
fresh human peripheral blood mononuclear cells (Sailybio, No. SLB-HP100A). The human pan
T cell was seeded into the 96-well plate and incubated at 37 °C under 5% CO2 for 1 hour. To the
cells 2µL of NECA and 2µL of test compound (final concentration of DMSO is 0.2%) were
added and the cells were incubated at 37 °C under 5% CO2 for 1 hour. T cells were treated by
adding 5µL of dynabeads of human T-activator CD3/CD28 (Gibcono. No.11131D) for T cell
expansion and activation and then incubated at 37°C under 5% CO2 for 1 hour. The plate was
centrifuged and the supernatant was transferred into another 96-well plate which was assayed for
IL-2 production using human IL-2 Quantikine ELISA Kit (R&D, No. D2050). Data were
collected and analyzed with Read Abs450 on envision.
5.3. Molecular docking
The crystal structure of hA2A AR in complex with antagonist ZM241385 (PDB ID: 5NM4)[41]
was downloaded from Protein Data Bank (http://www.pdb.org), and the structure of hA1 R in
complex with antagonist PSB36 (PDB ID: 5N2S)[41] was also retrieved. The crystal structures
of hA2A AR and hA1 AR were remodeled by first removing the antagonists from the protein and
second adding the missing residues and atoms using SWISS-MODEL.[42] Then the hydrogen
atoms and disulfide bonds of the proteins were added by AmerTools to prepare the initial
structures for molecular docking.
The molecular dockings were carried out by Schrodinger software (LLC, New York, NY, 2017).
The proteins and ligand preparations for docking were performed by Maestro (Schrödinger). The
proteins were prepared using the Protein Preparation Wizard, and the ligand was treated by
24

OPLS2005 force field. The induced fit Glide docking program[37] that considering the
conformational changes of the protein was used for docking studies. For the Glide docking, the
grid was defined using the auto-size box centered on residue Met365 of hA2A AR and Thr377 of
hA1 AR respectively. The protein preparation constrained refinement was selected, and the side-
chain trimming procedure as well as VDW scaling was used to create more room for the ligand
in the active site. The compound 27 was docked using Glide XP mode.[38,43] and the predicted
binding modes in hA2A AR and hA1 AR were ranked according to their glide scores.
Acknowledgements
Financial support from the National Natural Science Fundation of China (21772043, 82003651) is
greatly acknowledged. We also are thankful for the financial support from Guangdong
Innovative and Entrepreneurial Research Team Program (No. 2016ZT06Y337) and the
Fundamental Research Funds for the Central Universities (#19ykpy135, #20ykpy113,
No31143408)
Appendix. Supplementary data
Supplementary data associated with this article can be found in the online version.
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