molecules
Article2-Aryladenine Derivatives as a Potent Scaffold for Adenosine
Receptor Antagonists: The 6-Morpholino Derivatives
Filipe Areias 1,2,3,† , Carla Correia 1,† , Ashly Rocha 1 , Sofia Teixeira 1 , Marián Castro 2,4 , Jose Brea 2,4 ,Huabin Hu 5 , Jens Carlsson 5 , Maria I. Loza 2,4 , M. Fernanda Proença 1 and M. Alice Carvalho 1, *
1

2

3

4

5

*
†

Citation: Areias, F.; Correia, C.; Rocha,
A.; Teixeira, S.; Castro, M.; Brea, J.; Hu,
H.; Carlsson, J.; Loza, M.I.; Proença,
M.F.; et al. 2-Aryladenine Derivatives
as a Potent Scaffold for Adenosine
Receptor Antagonists: The

Centre of Chemistry of University of Minho (CQUM), Campus de Gualtar, Universidade do Minho,
4710-057 Braga, Portugal; filipeareias@yahoo.com.br (F.A.); carlabcpereira@gmail.com (C.C.);
ashlyrochaa@gmail.com (A.R.); id9191@alunos.uminho.pt (S.T.); fproenca@quimica.uminho.pt (M.F.P.)
Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago deCompostela, Avda de Barcelona, E-15782 Santiago de Compostela, Spain; marian.castro@usc.es (M.C.);
pepo.brea@usc.es (J.B.); mabel.loza@usc.es (M.I.L.)
School of Chemical Sciences & Engineering, Yachay Tech University, Yachay City of Knowledge,
Urcuqui 100119, Ecuador
Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Travesía da Choupana s/n,
E-15706 Santiago de Compostela, Spain
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University,
SE-75124 Uppsala, Sweden; huabin.hu@icm.uu.se (H.H.); jens.carlsson.lab@gmail.com (J.C.)
Correspondence: mac@quimica.uminho.pt
These authors contributed equally to this work.

Abstract: A set of 2-aryl-9-H or methyl-6-morpholinopurine derivatives were synthesized andassayed through radioligand binding tests at human A1 , A2A , A2B , and A3 adenosine receptor
subtypes. Eleven purines showed potent antagonism at A1 , A3 , dual A1 /A2A , A1 /A2B , or A1 /A3
adenosine receptors. Additionally, three compounds showed high affinity without selectivity for any
specific adenosine receptor. The structure-activity relationships were made for this group of newcompounds. The 9-methylpurine derivatives were generally less potent but more selective, and the
9H-purine derivatives were more potent but less selective. These compounds can be an importantsource of new biochemical tools and/or pharmacological drugs.
Keywords: G protein-coupled receptors; adenine derivatives; adenosine receptor antagonists;
2-arylpurine derivatives; structure-activity relationship

6-Morpholino Derivatives. Molecules
2024, 29, 2543. https://doi.org/
10.3390/molecules29112543

1. Introduction
Academic Editors: Igor Ivanov andZhangguo Gao
Received: 8 February 2024
Revised: 6 May 2024
Accepted: 24 May 2024
Published: 28 May 2024

Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Adenosine, a purine nucleoside composed of an adenine linked to a ribose via aβ-N9-glycosidic bond, modulates many physiological conditions related to neurological,
immunologic, and cardiovascular systems [1,2]. Adenosine receptors, namely A1 , A2A ,A2B , and A3 , belong to the G protein-coupled receptor (GPCR) superfamily and are widely
recognized as attractive targets for the design and development of new therapeutic agentsagainst different clinical disorders [3–5]. The activation of the A1 and A3 receptors inhibits
adenylyl cyclase activity through the action of Gi/o proteins and, as a result, intracellularcyclic AMP (cAMP) decreases, while the A2A and A2B receptors increase cAMP production
through the action of Gs proteins [6]. So, it is of therapeutic importance to synthesize new
drugs active on adenosine receptors A1 , A3 , and, additionally, dual A1 /A3 to study thesynergistic effect between these two receptor subtypes as they preferentially couple to thesame G proteins [2]. Indeed, there are many patents for adenosine receptor ligands, andsome of these ligands are already in clinical trials or waiting for FDA approval [4,5,7]. For
example, A1 ligands are useful for glaucoma, heart failure, angina, seizures, ischemia, depression, obesity, asthma, renal protection, edema associated with congestive heart failure,atrial arrhythmias, type II diabetes, neuropathic pain, neuroprotection, atrial fibrillation,

Molecules 2024, 29, 2543. https://doi.org/10.3390/molecules29112543

https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 2543

failure, atrial arrhythmias, type II diabetes, neuropathic pain, neuroprotection, abrillation, tachycardia, cardioprotection, and sleep regulation [1,8–12]. In addition
ands are useful for liver regeneration, hepatitis, psoriasis, rheumatoid arthritis, infl
tion, dry eye syndrome, fibrotic diseases, neurodegeneration, ischaemia,
asthma,2 of 17
obstructive pulmonary disease, glaucoma, and cancer [13–20], with A3 antagonist P(Palobiofarma SL), that has reached clinical trials for glaucoma, ulcerative colitis,
tachycardia, cardioprotection, and sleep regulation [1,8–12]. In addition, A3 ligands aresinophilic esophagitis [21,22]. Finally, dual A1/A3 ligands can be useful as potentia
useful for liver regeneration, hepatitis, psoriasis, rheumatoid arthritis, inflammation, dry
peutics
for treating
glaucoma,kidney failure,pulmonaryand Alzheim
eyesyndrome,
fibrotic diseases,
neurodegeneration,ischaemia,
asthma, diseases,
chronic obstructive
ease [23,24].
Despitethe factthat
severaladenosine
receptorPBF-677
ligands(Palobioare already inpulmonarydisease,glaucoma,andcancer
[13–20],with A3 antagonist
farma
SL),
that
has
reachedclinicaltrialsforglaucoma,
ulcerative
colitis,andeosinophilic
trials, achieving selectivity is still a challenge. Additionally, a number of side eﬀec
esophagitis [21,22]. Finally, dual A1 /A3 ligands can be useful as potential therapeutics forreported for A1 antagonists in the literature, such as an increase in the frequenc
treating glaucoma, kidney failure, pulmonary diseases, and Alzheimer’s disease [23,24].
zures, thestrokes[25,26],
nausea,transienthypertension,andachievtransient h
Despitefact thatseveraldizziness,
adenosine receptor
ligands
are alreadyin clinical trials,
sion
[27]
in
treatedpatients,which
justifies
thesearchfornewhigh-aﬃnity
ing selectivity is still a challenge. Additionally, a number of side effects were reported for A1 and s
antagonists
drugs. in the literature, such as an increase in the frequency of seizures, strokes [25,26],
dizziness,
nausea,transientwork,
hypertension,and transientand
hypotension[27] inpatients, for ad
In ourprevious
we synthesized
identified
a treated
new scaﬀold
which justifies the search for new high-affinity and selective drugs.
receptor
antagonists based on the adenine nucleus (Figure 1) [28]. Highly potent
In our previous work, we synthesized and identified a new scaffold for adenosine
lective compounds
wereonidentified
asnucleus
having(Figurea piperidinylgrouppotent
in C-6,a protonreceptor
antagonists based
the adenine
1) [28]. Highly
andand several
selectedarylgroupsin C-2 aofpiperidinyl
the purine
nucleus.
selective
compounds
wereidentified
as havinggroup
in C-6, aIndeed,
proton inthe
N-9,structur
andseveral selected(SAR)
aryl groups
in indicatedC-2 of the purine
structure-activity
ity relationshipstudythat nucleus.
an aryl Indeed,
unit atthe
C-2
is crucial for activit
relationship (SAR) study indicated that an aryl unit at C-2 is crucial for activity, and a
proton at N-9 increased potency. Furthermore, a piperidinyl group in C-6 instead
proton at N-9 increased potency. Furthermore, a piperidinyl group in C-6 instead of a 4-NN-methylpiperazinyl
led to withpurineswithhigheraffinity
or similar
for the ad
methylpiperazinylgroup group
led to purines
higheror similar
for theaﬃnity
adenosine
receptors
1 and
3 but
higherselectivity.
receptors
A1AandA3 A
but
withwithhigherselectivity.

H
N

5
6

N

2

Y
4
3

N

N
N
X

Figureadenosine
receptor
antagonists
scaffold identified
our previous
Figure1.1.TheThe
adenosine
receptor
antagonists
scaﬀoldinidentified
in work.
our previous work.

As an attempt to increase the affinity and selectivity of the compounds obtained in ourAsworkan attempt
to increase
the aﬃnity
andselectivityof thecompounds obta
previousand to establish
the importanceof group
X (Figure1), herein,
we synthesizednewpurinederivatives
substituted
with a protona methylgroup in 1),
N-9herein,our 6-morpholino
previous workandto establish
the importanceoforgroup
X (Figureanda selection
aryl substituents purine
in the C-2derivatives
position of thepurine nucleus.
thesized
newof6-morpholino
substituted
withThea results
proton or awill allow us to complete the SAR study for the adenine-based scaffold identified in ourgroup in N-9 and a selection of aryl substituents in the C-2 position of the purine nprevious publication.

The results will allow us to complete the SAR study for the adenine-based scaﬀo2.
Results
and previous
Discussionpublication.
tifiedin our
2.1. Chemistry

The target
3a–x were synthesized through the synthetic approach de2. Results
andcompounds
Discussion
scribed in Scheme 1. The starting reagents 1 [29] and 2 [28,30] were obtained following
2.1.procedures
Chemistrydescribed in previous work. Briefly, the commercially available diaminothemaleonitrile
was treatedwith triethyl
orthoformate,under reflux.
The solid
obtained
was appro
The target
compounds
3a-x
were synthesized
throughthesynthetic
reacted with the convenient amine followed by treatment with a base to generate imidascribed in Scheme 1. The starting reagents 1 [29] and 2 [28,30] were obtained follow
zoles 1. The compounds 2 were obtained in excellent yield through reaction of 1 with theprocedures
described
in previous
work.
Briefly, the commercially available diaminexcessof morpholine,
in acetonitrile,at room
temperature.

onitrile was treated with triethyl orthoformate, under reflux. The solid obtained
acted with the convenient amine followed by treatment with a base to generate imi
1. The compounds 2 were obtained in excellent yield through reaction of 1 with thof morpholine, in acetonitrile, at room temperature.
The products 3 were obtained through reaction of compounds 2 with diﬀere
hydes 4 following a previously described methodology [28,30]. The experimenta
tions used in each reaction mainly depended on the aldehyde. Generally, whenoles 1 were reacted with non-phenolic aldehydes 4, the reactions were performed

Molecules 2024, 29, 2543

celerate the reactions, most of them started at moderate temperature, but, when the TLC
showed the absence of imidazoles 2, the temperature was increased and the reactions weremonitored by TLC until the spots assigned to the intermediates were absent. In thesecases, the reactions led to black-greyish solids that required purification. The purification
3 of 17throughwas achieved through filtration of a dichloromethane solution of the compounds
a silica gel column.

Scheme1. Synthetic
approachderivatives
3a-x.
Scheme1. Synthetic
approachto
to 2-aryl-adenine
2-aryl-adenine derivatives
3a–x.

The products
3 were obtained
throughof compounds
2 with different
aldehyTo obtain
the phenolicderivatives,
thereaction
reactionswere performed
in an acidic
mediumdes 4 following a previously described methodology [28,30]. The experimental conditions
until complete consumption of the reagents (evidenced by TLC) and then continued in aused in each reaction mainly depended on the aldehyde. Generally, when imidazoles 1
basicwere
mediuma temperature
between4,40
80 °C.
In performed
these reactions,degradation ofreacted atwithnon-phenolic aldehydes
theandreactionswerein basic medium.
the reaction
mixturealso occurred
when higher temperatures
werealways
used. started
The pure
Consideringthat compounds
2 are temperature-sensitive,
the reactionsat productslow
wereobtainedtemperature.
following thepurification
procedure
described
for the other deto moderateWhenthe reactions
were carriedout underabove
those conditions(Method
A),
they
were
slower,but
theproducts
3
wereprecipitated
pure
from
solution
rivatives. The new compounds were fully characterized by usual techniques(1H and 13C
andisolatedthrough
simplefiltration
fromthereaction
medium.
Inorder
toaccelerate
NMR spectra are presented in the supporting information).
the reactions, most of them started at moderate temperature, but, when the TLC showed
the absence of imidazoles 2, the temperature was increased and the reactions were mon2.2. Pharmacology
itored by TLC until the spots assigned to the intermediates were absent. In these cases,
theled to synthesized
black-greyish insolidsrequired
Thegroup
purificationwas
Allreactions
compounds
thisthatwork
have purification.
a morpholine
as a substituent
achievedthroughfiltrationofadichloromethane
solutionofthecompounds
througha at C-2
at C-6, an H atom or a methyl group at N-9, and diﬀerent substituted aryl groups
silica gel column.
of the purine nucleus. The compounds (3a-x) were assessed through radioligand binding
To obtain the phenolic derivatives, the reactions were performed in an acidic medium
until complete consumption of the reagents (evidenced by TLC) and then continued ina basic medium at a temperature between 40 and 80 ◦ C. In these reactions, degradationof the reaction mixture also occurred when higher temperatures were used. The pureMolecules 2024, 29, 2543

4 of 17

products were obtained following the purification procedure described above for the otherderivatives. The new compounds were fully characterized by usual techniques (1 H and
13 C NMR spectra are presented in the supporting information).
2.2. Pharmacology
All compounds synthesized in this work have a morpholine group as a substituent at
C-6, an H atom or a methyl group at N-9, and different substituted aryl groups at C-2 of thepurine nucleus. The compounds (3a–x) were assessed through radioligand binding assays
at all human adenosine subtype receptors (A1 , A2A , A2B , and A3 ) expressed in mammaliancell lines. The percentage of inhibition (%inhib ) of radioligand binding was determined
for all compounds at 10 µM concentration. Those compounds showing a %inhib higher
than 80% were tested at different concentrations to determine their affinities (calculated
as pKi ) at the diverse receptors. Table 1 shows the binding affinities of all the synthesizedcompounds 3 at adenosine receptors A1 , A2A , A2B , and A3 .
Table 1. Binding affinities of compounds 3 at all human adenosine receptors expressed as %inhib at
10 µM or pKi . Values are reported as mean ± SEM of three experiments with duplicate measurements.

a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
t
u
v
x

R

R1

A1

A2A

A2B

A3

Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H

2-ClC6 H4
2-MeOC6 H4
2-HOC6 H4
3-ClC6 H4
3-HOC6 H4
4-ClC6 H42,3-(HO)2 C6 H3
2,5-Cl2 C6 H32,5-(HO)2 C6 H3
2-HO-5-MeOC6 H3
2-HO-5-ClC6 H3
2-F-5-MeOC6 H3
2,5-F2 C6 H3
2-HOC6 H4
3-ClC6 H4
3-HOC6 H4
4-ClC6 H4
3,4-Cl2 C6 H3
3-CF3 -4-ClC6 H3
2,5-Cl2 C6 H3
2-HO-5-ClC6 H3
2-F-5-MeOC6 H3
2,5-F2 C6 H3

36 ± 1%22 ± 1%
6.51 ± 0.11
6.80 ± 0.07
6.16 ± 0.21
37 ± 4%33 ± 0.3%
41 ± 2%
6.36 ± 0.15
6.28 ± 0.17
6.91 ± 0.08
61 ± 4%
6.28 ± 0.13
7.19 ± 0.10
7.75 ± 0.14
7.49 ± 0.06
7.02 ± 0.07
29 ± 1%43 ± 5%5.97 ± 0.11
7.05 ± 0.09
38 ± 2%
8.23 ± 0.06

11 ± 2%12 ± 5%
6.47 ± 0.05
15 ± 4%37 ± 1%42 ± 5%25 ± 2%2 ± 3%
6.25 ± 0.12
5.91 ± 0.11
6.15 ± 0.03
42 ± 1%37 ± 2%
6.97 ± 0.07
7.33 ± 0.07
6.26 ± 0.12
56 ± 3%22 ± 5%13 ± 5%44 ± 2%35 ± 1%45 ± 4%7.15 ± 0.05

22 ± 5%12 ± 2%5.70 ± 0.07
2 ± 2%
34 ± 3%15 ± 4%22 ± 1.5%
22 ± 3%
6.32 ± 0.01
5.78 ± 0.03
55 ± 1%27 ± 2%30 ± 4%
6.86 ± 0.10
7.12 ± 0.13
60 ± 6%50 ± 4%35 ± 3%8 ± 5%
26 ± 1%
6.21 ± 0.08
6.40 ± 0.16
7.44 ± 0.11

14 ± 2%36 ± 5%6 ± 5%
25 ± 4%52 ± 4%31 ± 5%42 ± 3%8 ± 1%
53 ± 5%19 ± 11%53 ± 4%5.28 ± 0.11
25 ± 3%
6.59 ± 0.06
7.40 ± 0.14
6.33 ± 0.08
7.47 ± 0.05
7.15 ± 0.08
6.02 ± 0.04
7.16 ± 0.17
37 ± 1%
7.83 ± 0.16
7.63 ± 0.12

Analysis of the data presented in the Table 1 shows that at 10 µM, several compounds
presented a percentage inhibition higher than 80%. The pKi was determined, being in
the range of 5.28 ≤ pKi ≤ 8.23 (3l, 3x). Both series, R = H and R = Me, gave ligands withhigh affinity to the four receptors subtypes; however, higher affinities are observed forderivatives with R = H (3d vs. 3h, 3c vs. 3n, 3e vs. 3p, 3k vs. 3u, 3l vs. 3v, and 3m vs. 3x).
The higher potency of derivatives with a proton at N-9 agrees with the results publishedin our previous work [28], but the increase in potency of N-9-methyl derivatives having
a morpholine unit at C-6 may indicate that the morpholine ring is interacting with thetarget through the oxygen atom. When we look for selectivity, we find selective ligands
either having a methyl group at N-9 (3d, 3e, 3l and 3m) or a proton (3p, 3r, 3s, 3t and 3v).
Additionally, the ligands with a methyl group at N-9 show high potency mainly for the
A1 receptor (3c, 3d, 3e, 3i, 3j, 3k, and 3m), and ligands with a proton at N-9 presented
high potency for both the A1 and A3 receptors (3n–3x). To evaluate the influence of theMolecules 2024, 29, 2543

5 of 17

R1 group on potency and selectivity against the four receptors, we analyzed first theR1 = chloro-derivatives, compounds 3a, 3d, 3f, 3h, 3o, 3q, 3r, 3s, and 3t.
In this set, the most potent and selective compound for the A1 receptor was 3d,with R1 = 3-ClC6 H4 [pKi (A1 ) = 6.80 ± 0.07]. Compounds 3r (R1 = 3,4-Cl2 C6 H3 ) and 3s(R1 = 3-CF3 -4-ClC6 H3 ) were highly potent and selective for the A3 receptor. Compound
3q (R1 = 4-ClC6 H4 ) showed dual selectivity for A1 /A3 receptors. These results seem toindicate that the presence of an electron-withdrawing chlorine atom at the meta position
together with a hydrophobic methyl group at N-9 interacts efficiently with the A1 receptor(3d). However, when the hydrophilic hydrogen was at N-9, a promiscuous ligand resulted(3o). The presence of a chlorine atom at the para position and a proton at N-9 interacts
efficiently with receptors A1 and A3 (3q). Compound 3q is a potential dual ligand forA1 /A3 receptors with therapeutic interest, as A1 and A3 receptors, principally coupled to
Gi/o proteins, can simultaneously inhibit the adenylyl cyclase activity [6,31,32].
In addition, selectivity was achieved when a second chlorine or a CF3 group was simultaneously at the meta position (3r, 3s), suggesting volume constraints at the A1 , A2A , andA2B pockets interacting with the ligands’ meta position. This hypothesis is also supportedby the lower pKi observed for compound 3s (R1 = 3-CF3 -4-Cl-C6 H3 ; pKi = 6.02 ± 0.04)
when compared to the pKi of compound 3r (R1 = 3,4-Cl2 C6 H3 ; pKi = 7.15 ± 0.08).
The sub-set of derivatives with hydroxyl groups at ortho or meta positions of theR1 group (3c, 3e, 3g, 3i, 3j, 3k, 3n, 3p, and 3u) will be analyzed. Generally, a hydroxyl
group at ortho or meta positions of R1 led to very potent but not selective ligands (3c,3i, 3j, 3k, 3n, and 3u), except compounds 3e and 3p, which have a hydroxyl groupat the meta position of R1 and showed selectivity for the A1 receptor. Derivative 3d(R1 = 3-ClC6 H4 ) also showed selectivity for the A1 receptor. These results suggest that anelectron-withdrawing group produced through the inductive effect at the meta positionof R1 interacts efficiently with a polar moiety at the A1 receptor. Comparing the affinity
values between 3b (R1 = 2-MeOC6 H4 ) and 3c (R1 = 2-HOC6 H4 ), the increase of affinityof 3c suggests that a hydrogen-bond donor (HBD) group at the ortho position of R1 isimportant for the interaction with the adenosine receptors A1 , A2A , and A2B . However,the possibility of a hydrogen-bond-acceptor (HBA) interaction of the ligand with the target should also be considered. Hydroxyl and methoxyl groups may interact that way
with the targets; however, if the receptor pockets have volume constraints, this will explain the lack of activity of derivative 3b and support the hypothesis of HBD interaction.
Furthermore, compound 3c has almost the same submicromolar affinity for the A1 andA2A receptors. This result indicates that 3c is a dual ligand for A1 /A2A receptors. Dualaffinity for the A1 /A2A receptors was also observed for ligand 2k. These ligands could
have medicinal interest, as compounds with dual affinity at A1 /A2A receptors have shown
therapeutic efficacy in animal models of Parkinson’s disease [33,34]. On the other hand,compound 3u showed a dual affinity for receptors A1 /A2B . A comparison of affinities
registered for 3c, 3d, and 3e with 3n, 3o, and 3p suggests more hydrophilic or small pockets at receptors to accommodate the group at N-9 of the ligands. Additionally, compound 3e(R1 = 3-HOC6 H4 ) is a very potent and selective ligand for the A1 receptor with
pKi = 6.16 ± 0.21, while compound 3g (R1 = 2,3-(HO)2 C6 H3 ) has a low affinity for allof the receptors. This may indicate that an intramolecular hydrogen bond between
the hydroxyl groups of the ligand precludes the interaction with the target, leading to
low activity.
Finally, the fluoro derivatives (3l, 3m, 3v, and 3x) presented high potency and selectivity for receptors A1 and A3 , except compound 3x, which showed no selectivity.
Compounds 3l and 3v with R1 = 2-F-5-MeOC6 H3 were selective to the A3 receptor, with
pKi = 5.28 ± 0.11 and pKi = 7.83 ± 0.16, respectively. Compound 3v also showed a highaffinity for receptor A2B (pKi = 6.40 ± 0.16). These results suggest volume restrictions in
the pockets of receptors A2B and A3 to lodge the methyl group at N-9 of the ligand, withthe available space in receptor A3 bigger than in receptor A2B . Compound 3m showed highaffinity and selectivity for the A1 receptor, while compound 3x showed higher potency

Molecules 2024, 29, 2543

6 of 17

Molecules 2024, 29, x FOR PEER REVIEW
6 of 18
for all receptors but no selectivity. These results also support the hypothesis that there
Molecules 2024, 29, x FOR PEER REVIEW
6 of 18is

limited space in the receptors’ pockets to accommodate bulky groups at N-9. The differencein the selectivity of compounds 3l and 3m may indicate that receptor A3 has a hydrophobic
receptor
A1 does not. This
also supported
results
obtained
forligand,ligands
pocketto accommodate
thehypothesis
substituentisMeO
present at bythethemeta
positionof thereceptorA
1 does not. This hypothesis is also supported by the results obtained for ligands
3v and3x. Figure
2 represents
SAR modelisthat
thetheresults.
while
receptor
A doesnot. Thisahypothesis
alsosummarizes
supported byresults obtained for3v and 3x. Figure 12 represents a SAR model that summarizes the results.
ligands 3v and 3x. Figure 2 represents a SAR model that summarizes the results.
- Selective ligand antagonists for adenosine receptors- Selective ligand antagonists for adenosine receptors
A3
RY
R M e Y 2-F-5-M eO
M eH 2-F-5-M
eO eO
2-F-5-M
H
2-F-5-MeO
2,5-Cl2
2,5-Cl2
3,4-Cl2
3,4-Cl
2 3 -4-Cl
3-CF
3-CF 3-4-Cl

A1
RY
R M e Y 3-Cl
M e 3-Cl3-HO
3-HO
2,5-F 22
H 2,5-F
3-HO
H
3-HO- Dual affinity- Dualaffinity
antagonists
antagonists

Y

A3

A1

A 1 /A 2A
A 1 /A 2A
A 1 /A 2B
A 1 /A 2B
A 1 /A 3
A 1 /A 3

R
R

2

N
N

N

2

N

3 4
5
6
5
6

N

N

N

N

N

Y3 4

R1

R1

N
O
O

Y
Y
M e 2-HO-5-Cl
M e 2-HO-5-Cl
H
2-HO-5-Cl
H
2-HO-5-Cl
H
4-Cl
H
4-Cl
R

R

FigureStructure–activity
relationship
2-aryl-9-substituted-6-morpholino
purinederivatives
Figure2. 2.
Structure-activity
relationshipof of
2-aryl-9-substituted-6-morpholino
purinederivatives
forforFigure2. Structure–activity
relationship of 2-aryl-9-substituted-6-morpholino purine derivatives fortheadenosine
receptor
antagonists.
the adenosine receptor antagonists.
the adenosine receptor antagonists.

The
mostpotent
potentcompounds
compoundsfor
forAA11 (3x;(3x; pK
0.06;
Figure3a)3a)
andandA3 (3v;
pKi =
The
most
pKii ==8.23
8.23± ±
0.06;
FigureA3 (3v;
The± mostpotentcompounds
for Aand1 (3x; pKi = 8.23 ± 0.06; Figure 3a) and A3 (3v; pKi =0.16)
receptors
wereselected
testedinintracellular
cAMP
tests
to
study
their
pK7.83
=
7.83
±
0.16)
receptors
wereselected
andtestedinintracellular
cAMP
tests
to
study
i
7.83functional± 0.16) receptors
wereselected and testedbehavior
in intracellularcAMP
tests to study
theirwasactivity.
Theagonist/antagonist
of
the
two
compounds
selected
their functional activity. The agonist/antagonist behavior of the two compounds selectedfunctional
activity.
The agonist/antagonist
behaviorof the twocompounds
selectedwasdeterminedbybyassessingof cAMPcAMP
levelsbybythereceptor
receptorwasdeterminedassessingtheir
theireﬀect
effectonon thethe modulation oflevelsthedeterminedby assessing
theireﬀecttheonresult
the modulationof cAMPtest
levels theby thereceptorpoagonist
NECA.
Figure3b shows
of a representative
antagonist
agonistNECA.
Figure3b shows
the resultof a representative
test for thefor
antagonist
potencyagonistNECA.
Figure3bshows
theresultofarepresentative
test
for
the
antagonist
poof compoundat A1 receptor.
The results
confirmed
thatcompounds
the compounds
3v
and
oftency
compound3x at A13xreceptor.
The results
confirmedthat the3v and3x
tency
ofcompound3x
atA
1 receptor. The results confirmed that the compounds 3v andare antagonistsof the3 and
1 receptors,andandtheirtheirantagonist
antagonist potencies, expressed
are3xantagonistsof theA3 A
andA1Areceptors,
expressedas
3xof8.24the
3 and A1 receptors, and their antagonist potencies, expressed as
pK
were±A0.11and and8.25 8.25± 0.16,(Table (Table2). Based
their onstructuralasare
pKBantagonists
values,were
8.24± 0.11± respectively
0.16, respectively2). onBasedtheir
Bvalues,pK
B values, were 8.24 ± 0.11 and 8.25 ± 0.16, respectively (Table 2). Based on their structural
structuralsimilarity,
we can generalizethat all compoundsof thisare antagonistsofsimilarity,
we can generalizethat all compoundsof this work
arework
antagonistsof adenosine
similarity,
wecangeneralizethatallcompounds
of
this
work
are
antagonistsof
adenosine
adenosine
receptorsreceptors.
.
receptors.
(a)

(a)

(b)
(b)

Figure 3. (a) Competition binding curve for compound 3x at A1 receptors. (b) Concentration-response
curve
of compound
3x in the binding
presencecurveof 10 µM
of NECA at3x
human
expressed
in CHO
Figure3. (a) Competition
for compoundat A1 Areceptors.
Concentration-re1 receptor(b)Figure3. (a)
Competition
binding
curve
fordeviation
compound3xofatNECA
A
1 receptors.
(b)AConcentration-recells.
Pointsrepresent
the meanbars)
ofmeasurements.
sponsecurveof compound
3x±instandard
the presenceof 10(verticalµM
atduplicate
human
1 receptor expressedsponsecurveof compound
3x in theof 10 µMdeviationof NECA(verticalat humanA1 receptor
expressed
in CHO
cells.
Points represent
thepresence
mean ± standard
bars)
of duplicate
measurein CHO
cells. Points represent the mean ± standard deviation (vertical bars) of duplicate measurements.
ments.

Molecules 2024, 29, x FOR PEER REVIEW
Molecules 2024, 29, 2543

7 of 18
7 of 17

Table 2. Antagonist potency (pKB) of the most potent compounds at human A1 and A3 adenosine
Table
2. Antagonist
potency
the most
potentat human
A1experiments
and A3 adenosineB ) ofare
receptors
in cAMP
tests. (pK
Valuesreportedascompounds
mean ± SEM
of threewith receptors
duplicateinmeasurements.
cAMP tests. Values are reported as mean ± SEM of three experiments with duplicate measurements.
Compounds
Compounds
3v3v
3x3x

PotencyB ) B)
Potency(pK
(pK
AA
1 1

N.D.
N.D.
8.25
8.25±±0.16
0.16

AA
3 3
8.24
0.11
8.24 ±±0.11
N.D.
N.D.

N.D.—Not
determined.
N.D.—Not
determined.

InInthiswewe
synthesized
highlypotentandandselective
hitshits
basedon theforthiswork,work,
synthesized
highlypotentselective
based
onscaffold
the scaﬀold
adenosine
receptor
antagonists
identified
in ourpublication[28]. These
results
for adenosine
receptor
antagonists
identified
inprevious
our previous
publication[28]. These
reindicatethatthepurines
substituted
withamorpholine
group
at
C-6,
a
hydrogenatomorasults indicate that the purines substituted with a morpholine group at C-6, a hydrogen
methyl
group
at
N-9,
and
a
specific
aryl
group
at
C-2
of
purinenucleus,gave
highaffinity
atom or a methyl group at N-9, and a specific aryl group at C-2 of purine nucleus, gave
andselective
forantagonists
adenosine for
A1 adenosineand A3 receptors,
as3 wellas dualhighaﬃnityantagonists
and selective
A1 and A
receptors,
asantagonists
well as dual
forreceptors A
and. In A
addition,four compounds(3i, 3j, 3n,(3i,and
1 /A
2A , A1 /A
1 /A
antagonists
forreceptors
A12B/A, 2A
, AA
1/A
2B, 3
and
1/A3. In addition,
four compounds
3j,
3o)affinity,
althoughthey wereselective
for any
adenosine
3n,showed
and 3o) highshowedhigh aﬃnity,
althoughtheynot
were
not selective
forspecificany specificadenoreceptor
subtype.
sine receptor
subtype.
2.3. Docking Studies
2.3. Docking Studies
Compound 3x was computationally docked to the orthosteric site of the A1 recepCompound 3x was computationally docked to the orthosteric site of the A1 receptor
tor using a crystal structure representing an inactive conformation (PDB accession code:using a crystal structure representing an inactive conformation (PDB accession code:
5UEN) [35]. The ligand was predicted to form hydrogen bonds with Asn254 and π-π stack5UEN) [35]. The ligand was predicted to form hydrogen bonds with Asn254 and π-πing with Phe171, which are interactions characteristic of adenosine receptor antagonists
stacking with Phe171, which are interactions characteristic of adenosine receptor antago(Figure 4) [36]. The predicted binding mode of compound 3x in the A1 receptor also exnists (Figure 4) [36]. The predicted binding mode of compound 3x in the A1 receptor also
plained observed SARs. For example, replacing the fluorine at the 5-position of the phenyl
explained observed SARs. For example, replacing the fluorine at the 5-position of the phering with a methoxy group led to a substantial loss of affinity at the A1 receptor (pKi < 5,nyl ring with a methoxy group led to a substantial loss of aﬃnity at the A1 receptor (pKi <compound 3v). Consistent with this observation, increasing the size of this substituent in
5,3v).
Consistent
withthis withobservation,
our compoundmodel of thecomplex
led toclashes
Asn184 increasingand His251.the size of this substituent
in our model of the complex led to clashes with Asn184 and His251.

Figure4.4.Predicted
Predictedbinding
bindingmodemodeofofcompoundcompound3x3xininthe
theorthostericorthostericsite
siteofofthe
the1 receptor. The
FigureA1Areceptor.
The
receptorisdepictedasagrey
cartoon,withtheligandandkey
sidechainsrepresented
sticks.
receptor is depicted as a grey cartoon, with the ligand and key side chains represented asassticks.
Hydrogen bonds are shown as black dashed lines.
Hydrogen bonds are shown as black dashed lines.

Molecules 2024, 29, 2543

8 of 17

3. Experimental Protocols
3.1. Chemistry
The imidazoles 1 used in this work were synthesized according to previously described
procedures [29], and compounds 2 were synthesized according to the procedure describedin [28,30]. Solvents and other commercially available chemicals were used as shipped. The
melting points (m.p.) were determined with a Gallenkamp melting point apparatus, andthey are uncorrected. The reactions were followed by thin layer chromatography (TLC)
using Silica Gel 60 F254 (Merck, Darmstadt, Germany) plates with detection by UV light.
The NMR spectra were obtained on a Varian Unity Plus (1 H: 300 MHz, 13 C: 75 MHz) oron a Bruker Avance III NMR spectrometer (1 H: 400 MHz, 13 C: 100 MHz) including the 1 H
and 13 C bidimensional correlation spectra (HMQC and HMBC). For solutions, [D6 ]-DMSO
residual [D6 ]-DMSO (δH = 2.49 ppm) or [D6 ]-DMSO (δC = 39.5 ppm) was used as theinternal standard at 298 K. Chemical shifts (δ) were reported in parts per million (ppm) andthe coupling constants, J, were presented in hertz (Hz). The purity of all tested compounds
was higher than 95% according to elemental analysis, which was reported to be within
0.4% of the calculated values. The IR spectra were recorded with a FT-IR Bomem MB
104 using nujol mulls and NaCl cells. Elemental analyses were performed with a LECO
CHNS-932 instrument.
3.2. General Procedure for the Synthesis of 3a–x
Method A: To a suspension of imidazole 2 in ethanol, an ethanol/acetonitrile mixture,
or DMSO, the aldehyde 4 (1.1–1.5 equivalents) was added, followed by triethylamine(10 equivalents). The reaction was kept at room to moderate temperature and followed
by TLC. Otherwise, when the TLC indicated the absence of the starting reagent but thepresence of intermediates, the reaction continued at a higher temperature. When the
TLC showed the absence of intermediate spots, the solvent was removed in the rotary
evaporator, and an off-white solid was isolated after the addition of a small amount of
ethanol to the residue. When the residue showed a dark color, it was dissolved in DCM.
The solution was filtered through a column of silica gel (0.5 cm high), and the column waswashed with an extra 30 mL of DCM. The resulting solution was concentrated until dry,
diethyl ether was added to the oil, and an off-white solid was precipitated. The solid wasfiltered and washed with cold diethyl ether.
Method B: To a suspension of imidazole 2 in ethanol, the aldehyde 4 (1.1–1.5 equivalents) was added, followed by trifluoroacetic acid (1.3–2.0 equivalents). The reaction
was kept at 22 ◦ C under a magnetic stirrer until the TLC showed consumption of starting
reagent 2. The solvent was eliminated in the rotary evaporator, dimethyl sulfoxide and/or
ethanol was added, followed by triethylamine (10 equivalents), and the reaction continued
at 40–80 ◦ C. When the TLC indicated the end of the reaction, the solvents were removed in
the rotary evaporator, and the product was precipitated through the addition of water. The
isolated solid was dissolved in a mixture of DCM/THF/EtOH and purified through dry
flash chromatography using DCM as the elution solvent.
3.2.1. 4-(2-(2-Chlorophenyl)-9-methyl-9H-purin-6-yl)morpholine 3a
Method A: Compound 2a (0.18 g, 0.89 mmol) in EtOH, aldehyde 4a (1.1 eq.), andEt3 N (10 eq.) was kept at 22 ◦ C for 20 days. Product 3a was isolated as an off-white solid(0.12 g, 0.28 mmol, 31%); m.p. = 99–101 ◦ C; Found: C, 58.28; H, 4.86; N, 21.25. C16 H16 ClN5 O
requires C, 58.27; H, 4.89; N, 21.24; IR (Nujol mull) νmax: 1584 cm−1 ; 1 H (300 MHz, DMSOd6) δ: 8.21 (s, 1H), 7.75–7.72 (m, 1H), 7.53–7.50 (m, 1H), 7.45–7.38 (m, 2H), 4.23 (br s, 4H),
3.75 (s, 3H), 3.71 (t, J = 4.5 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ: 157.6, 152.7, 151.6, 141.4,
138.7, 131.6, 131.5, 130.1, 129.9, 126.8, 117.7, 66.2, 45.2, 29.6.
3.2.2. 4-(2-(2-Methoxyphenyl)-9-methyl-9H-purin-6-yl)morpholine 3b
Method A: Compound 2a (0.13 g, 0.64 mmol) in DMSO, aldehyde 4b (1.1 eq.), andEt3 N (10 eq.) was kept at 60 ◦ C for 6 days. Product 3b was isolated as an off-whiteMolecules 2024, 29, 2543

9 of 17solid (0.05 g, 0.23 mmol, 36%); m.p. = 114–116 ◦ C; Found: C, 62.10; H, 5.92; N, 21.15.
C17 H19 N5 O2 .0.2H2 O requires C, 62.08; H, 5.90; N, 21.30; IR (Nujol mull) νmax: 1698, 1587,
1577 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 8.15 (s, 1H), 7.50 (dd, J = 7.7 Hz, J = 1.8 Hz, 1H),
7.37 (td, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 4.20 (br s,
4H), 3.80–3.50 (m, 10H). 13 C (75 MHz, DMSO-d6) δ: 158.4, 157.2, 152.8, 151.7, 149.9, 130.6,
130.1, 129.7, 120.0, 112.5, 66.2, 55.9, 45.2, 29.5.
3.2.3. 2-(9-Methyl-6-morpholino-9H-purin-2-yl)phenol 3c
Method A: Compound 2a (0.08 g, 0.38 mmol) in a mixture of EtOH/CH3 CN (1/1)
and aldehyde 4c (1.5 eq.) was kept at 11 ◦ C for 17 days. Product 3c was isolated as an
off-white solid (0.08 g, 0.25 mmol, 66%); m.p. = 210–212 ◦ C (Found: C, 61.17; H, 5.52; N,
21.00. C16 H17 N5 O2 .0.1H2 O requires C, 61.45; H, 5.49; N, 22.40); IR (Nujol mull) νmax: 3097,
1591, 1530, 1519 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.52 (s, 1H, OH), 8.38 (dd, J = 8.4 Hz,
J = 1.8 Hz, 1H), 8.21 (s, 1H), 7.33 (td, J = 7.8 Hz, J = 1.8 Hz, 1H), 6.91 (t, J = 7.8 Hz, 1H), 6.89
(d, J = 7.8 Hz, 1H), 4.25 (br s, 4H), 3.78 (s, 4H), 3.77 (s, 3H); 13 C (75 MHz, DMSO-d6) δ: 159.4,
157.5, 152.4, 149.7, 141.4, 132.0, 129.0, 119.3, 118.6, 117.8, 117.2, 66.1, 45.4, 29.6.
3.2.4. 4-(2-(3-Chlorophenyl)-9-methyl-9H-purin-6-yl)morpholine 3d
Method A: Compound 2a (0.31 g, 1.46 mmol) in EtOH, aldehyde 4d (1.1 eq.), andEt3 N (10 eq.) was kept at 20 ◦ C for 12 days and then at 70 ◦ C for 21 h. The product 3d was
isolated as an off-white solid (0.35 g, 1.06 mmol, 73%); m.p. = 160–162 ◦ C (Found: C, 58.04;
H, 4.76; N, 21.18. C16 H16 ClN5 O requires C, 58.27; H, 4.89; N, 21.24); IR (Nujol mull) νmax:
1690, 1575 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 8.31 (m, 2H), 8.17 (s, 1H), 7.48–7.50 (m, 2H),
4.26 (br s, 4H), 3.76 (br s, 4H), 3.73 (s, 3H); 13 C (75 MHz, DMSO-d6) δ: 155.3, 153.0, 151.9,
141.5, 140.4, 133.2, 130.2, 129.6, 127.1, 126.3, 118.4, 66.2, 45.3, 29.5.
3.2.5. 3-(9-Methyl-6-morpholino-9H-purin-2-yl)phenol 3e
Method B: Compound 2b (0.22 g, 1.10 mmol) in EtOH, aldehyde 4e (1.1 eq.), and TFA(2 eq.) was kept at 22 ◦ C for 3 days. Then, the reaction continued in DMSO and Et3 N(10 eq.) at 60 ◦ C for 2 days. The product 3e was isolated as an off-white solid (0.23 g,
0.75 mmol, 67%); m.p. = 230–232 ◦ C (Found: C, 61.67; H, 5.52; N, 22.64. C16 H17 N5 O2
requires C, 61.72; H, 5.50; N, 22.49); IR (Nujol mull) νmax: 3281, 1578, 1505 cm−1 ; 1 H(300 MHz, DMSO-d6) δ: 9.45 (s, 1H, OH), 8.16 (s, 1H), 7.84 (br.s, 2H), 7.24 (t, J = 8.4 Hz, 1H),
6.82 (d, J = 7.8 Hz, 1H), 4.28 (br s, 4H), 3.77 (s, 3H), 3.75 (m, 4H); 13 C (75 MHz, DMSO-d6) δ:
157.3, 156.8, 152.9, 152.0, 141.1, 139.7, 129.1, 118.6, 118.1, 116.7, 114.5, 66.2, 45.1, 29.4.
3.2.6. 4-(2-(4-Chlorophenyl)-9-methyl-9H-purin-6-yl)morpholine 3f
Method A: Compound 2a (0.34 g, 1.57 mmol) in EtOH, aldehyde 4f (1.1 eq.), and Et3 N(10 eq.) was kept at 21 ◦ C for 8 days and then at 70 ◦ C for 6 h. The product 3f was isolated
as an off-white solid (0.25 g, 0.75 mmol, 48%); m.p. = 226–228 ◦ C (Found: C, 57.99; H, 4.86;
N, 21.13. C16 H16 ClN5 O requires: C, 58.27; H, 4.89; N, 21.24); IR (Nujol mull) νmax: 1706,
1587, 1571 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 8.40 (d, J = 8.4 Hz, 2H), 8.17 (s, 1H), 7.52 (d,J = 8.4 Hz, 2H), 4.28 (br s, 4H), 3.79 (s, 3H), 3.74 (m, 4H); 13 C (75 MHz, DMSO-d6) δ: 155.7,
152.9, 151.0, 141.4, 137.1, 134.5, 129.4, 128.3, 118.2, 66.2, 45.1, 29.5.
3.2.7. 3-(9-Methyl-6-morpholino-9H-purin-2-yl)benzene-1,2-diol 3g
Method B: Compound 2a (0.15 g, 0.69 mmol) in EtOH, aldehyde 4g (1.1 eq.), andTFA (2 eq.) was kept at 22 ◦ C for 3 days. Then, the reaction continued in a mixture of
DMSO/EtOH (0.1/1) and Et3 N (10 eq.) at 80 ◦ C for 6 h. The product 3g was isolated as
an off-white solid (0.14 g, 0.43 mmol, 62%); m.p. = 254–256 ◦ C (Found: C, 58.87; H, 5.55;
N, 21.02. C16 H17 N5 O3 C16H17N5O3 requires C, 58.71; H, 5.23; N, 21.39); IR (Nujol mull)νmax: 3415, 1588, 1569 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.73 (s, 1H, OH), 8.83 (br s, 1H,
OH), 8.19 (s, 1H), 7.83 (dd, J = 8.0 Hz, J = 1.5 Hz, 1H), 6.84 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H),

Molecules 2024, 29, 2543

10 of 176.70 (t, J = 7.8 Hz, 1H), 4.23 (br s, 4H), 3.77 t, J = 5.4 Hz, 7H); 13 C (75 MHz, DMSO-d6) δ:
157.9, 152.3, 149.8, 148.3, 146.1, 141.3, 119.5, 119.1, 117.9, 117.6, 117.3, 66.0, 45.4.
3.2.8. 4-(2-(2,5-Dichlorophenyl)-9-methyl-9H-purin-6-yl)morpholine 3h
Method A: Compound 2a (0.25 g, 1.22 mmol) in EtOH, aldehyde 4h (1.1 eq.), and Et3 N(10 eq.) was kept at 40 ◦ C for 4 days and then at 50 ◦ C for 10 days. The product 3h wasisolated as an off-white solid (0.18 g, 0.50 mmol, 41%); m.p. = 141–142 ◦ C (Found: C, 52.64;
H, 4.11; N, 19.45. C16 H15 Cl2 N5 O requires C, 52.76; H, 4.15; N, 19.23); IR (Nujol mull) νmax:
3048, 1585 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 8.22 (s, 1H), 7.78(d, J = 2.7 Hz, 1H), 7.56 (d,J = 8.4 Hz, 1H), 7.50 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 4.23 (br s, 4H), 3.76 (s, 3H), 3.72 (t,J = 4.5 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ: 156.3, 152.7, 151.5, 141.6, 140.0, 131.9, 131.4,
130.9, 130.5, 129.7, 117.9, 66.2, 45.2, 29.6.
3.2.9. 2-(9-Methyl-6-morpholino-9H-purin-2-yl)benzene-1,4-diol 3i
Method B: Compound 2a (0.20 g, 0.96 mmol) in EtOH, aldehyde 4i (1.1 eq.), and TFA(2 eq.) was kept at 22 ◦ C for 5 h. Then, the reaction continued in a mixture of DMSO/EtOH(0.1/1) and Et3 N (10 eq.) at 60 ◦ C for 3 days. The product 3i was isolated as an off-whitesolid (0.20 g, 0.61 mmol, 64%); m.p. = 288–290 ◦ C (Found: C, 57.21; H, 5.09; N, 20.61.
C16 H17 N5 O3 .0.5H2 O requires C, 57.14; H, 5.36; N, 20.83); IR (Nujol mull) νmax: 3353, 1574,
1500 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 12.81 (s, 1H, OH), 8.88 (s, 1H, OH), 8.20 (s, 1H),
7.80 (d, J = 2.7 Hz, 1H), 6.77 (dd, J = 8.6 Hz, J = 2.7 Hz, 1H), 6.72 (d, J = 8.7 Hz, 1H), 4.25 (brs, 4H), 3.77 (t, J = 3.6 Hz, 7H); 13 C (75 MHz, DMSO-d6) δ: 157.6, 152.4, 152.2, 149.9, 149.2,
141.4, 119.8, 119.3, 117.7 (2C), 114.1, 66.1, 45.4, 29.7.
3.2.10. 4-Methoxy-2-(9-methyl-6-morpholino-9H-purin-2-yl)phenol 3j
Method B: Compound 2a (0.15 g, 0.70 mmol) in EtOH, aldehyde 4j (1.2 eq.), and TFA(2 eq.) was kept at 22 ◦ C for 2 days. Then, the reaction continued in EtOH and Et3 N (10 eq.)at 25 ◦ C for 5 days. The product 3j was isolated as an off-white solid (0.14 g, 0.43 mmol,62%); m.p. = 146–148 ◦ C (Found: C, 59.75; H, 5.67; N, 20.42. C17 H19 N5 O3 requires C, 59.81;
H, 5.61; N, 20.52); IR (Nujol mull) νmax: 3011, 1594, 1578 cm−1 ; 1 H (300 MHz, DMSO-d6) δ:
12.99 (s, 1H, OH), 8.20 (s, 1H), 7.89 (d, J = 3.2 Hz, 1H), 6.97 (dd, J = 8.8 Hz, J = 3.2 Hz, 1H),
6.83 (d, J = 8.8 Hz, 1H), 4.23 (s, 4H), 377 (m, 7H); 13 C (75 MHz, DMSO-d6) δ: 157.2, 153.4,
152.4, 151.5, 149.8, 141.4, 119.3, 118.7, 117.9, 117.8, 112.6, 66.0, 55.4, 45.4, 29.6.
3.2.11. 4-Chloro-2-(9-methyl-6-morpholino-9H-purin-2-yl)phenol 3k
Method B: Compound 2a (0.13 g, 0.64 mmol) in EtOH, aldehyde 4k (1.1 eq.), andTFA (2 eq.) was kept at 22 ◦ C for 2 days. Then, the reaction continued in a mixture of
DMSO/EtOH (0.2/1) and Et3 N (10 eq.) at 60 ◦ C for 6 days. The product 3k was isolated as
an off-white solid (0.104 g, 0.41 mmol, 64%); m.p. = 179–180 ◦ C (Found: C, 55.65; H, 4.50;
N, 20.00. C16 H16 ClN5 O2 requires C, 55.58; H, 4.66; N, 20.25); IR (Nujol mull) νmax: 3016,
1589, 1528 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.59 (s, 1H, OH), 8.24 (d, J = 2.8 Hz, 1H),
8.20 (s, 1H), 7.32 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 4.20 (s, 4H), 3.77 (t,J = 4.8 Hz, 7H); 13 C (75 MHz, DMSO-d6) δ: 158.0, 156.0, 152.2, 149.5, 141.5, 131.4, 127.7,
122.2, 120.5, 119.1, 118.0, 66.0, 45.3, 29.6.
3.2.12. 4-(2-(2-Fluoro-5-methoxyphenyl)-9-methyl-9H-purin-6-yl)morpholine 3l
Method A: Compound 2a (0.16 g, 0.77 mmol) in EtOH, aldehyde 4l (1.3 eq.), and Et3 N(10 eq.) was kept at 22 ◦ C for 1 day and then at 60 ◦ C for 4 days. The product 3l was
isolated as an off-white solid (0.15 g, 0.44mmol, 57%); m.p. = 102–104 ◦ C (Found: C, 58.76;
H, 4.93; N, 19.99. C17 H18 FN5 O2 ·0.2H2 O requires C, 58.86; H, 5.31; N, 20.19); IR (Nujol mull)νmax: 3122, 1598, 1579 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 8.19 (s, 1H), 7.52 (dd, J = 3.2 Hz,
J = 6.4 Hz, 1H), 7.18 (dd, J = 8.0 Hz, J = 6.9 Hz, 1H), 7.01 (dt, J = 6.9 Hz, J = 2.4 Hz, 1H),
4.23 (br s, 4H), 3.80–3.70 (m, 10H). 13 C (75 MHz, DMSO-d6) δ: 155.2 (d, J = 4 Hz), 154.9

Molecules 2024, 29, 2543

11 of 17(d, J = 2 Hz), 154.7 (d, J = 244 Hz), 153.4, 151.7, 141.4, 127.7 (d, J = 11 Hz), 117.8, 117.3 (d,J = 24 Hz), 115.9 (d, J = 2 Hz), 115.8 (d, J = 8 Hz), 66.2, 55.6, 45.2, 29.5.
3.2.13. 4-(2-(2,5-Difluorophenyl)-9-methyl-9H-purin-6-yl)morpholine 3m
Method A: Compound 2a (0.31 g, 1.48 mmol) in EtOH, aldehyde 4m (1.1 eq.), andEt3 N (10 eq.) was kept at 40 ◦ C for 4 days and then at 50 ◦ C for 6 days. The product 3m was
isolated as an off-white solid (0.30 g, 0.91 mmol, 61%); m.p. = 143–145 ◦ C (Found: C, 57.90;
H, 4.77; N, 20.97. C16 H15 F2 N5 O requires C, 58.00; H, 4.56; N, 21.14); IR (Nujol mull) νmax:
3083, 1616, 1578 cm−1 ; 1 H (400 MHz, DMSO-d6) δ: 8.20 (s, 1H), 7.80–7.79 (m, 1H), 7.34–7.30(m, 1H), 4.24 (br s, 4H), 3.77 (s, 3H), 3.73 (t, J = 4.5 Hz, 4H); 13 C (100 MHz, DMSO-d6)
δ: 157.7 (dd, J = 230 Hz, J = 6 Hz), 156.5 (dd, J = 246 Hz, J = 4 Hz), 154.2 (dd, J = 5 Hz,
J = 2 Hz), 152.9, 151.6, 141.6, 128.5 (dd, J = 11 Hz, J = 4 Hz), 118.4 (dd, J = 25 Hz, J = 9 Hz),118.0, 117.5 (dd, J = 24 Hz, J = 9 Hz), 128.3 (dd, J = 25 Hz, J = 2 Hz), 66.2, 45.2, 29.5.
3.2.14. 2-(6-Morpholino-9H-purin-2-yl)phenol 3nMethod B: Compound 2b (0.30 g, 1.55 mmol) in EtOH, aldehyde 4c (1.1 eq.), and TFA(1.5 eq.) was kept at 22 ◦ C for 4.5 h. Then, the reaction continued in DMSO and Et3 N(10 eq.) at 40 ◦ C for 10 days. The product 3n was isolated as an off-white solid (0.15 g,
0.52 mmol, 34%); (Found: C, 60.43; H, 5.12; N, 23.71. C15 H15 N5 O2 requires C, 60.60; H, 5.09;
N, 23.56); 1 H (400 MHz, DMSO-d6) δ: 13.64 (s, 1H), 13.23 (br s, 1H), 8.35 (dd, J = 8.4 Hz,
J = 2.0 Hz, 1H), 8.20 (s, 1H), 7.31 (dt, J = 8.4 Hz, J = 2.0 Hz, 1H), 6.90 (m, 2H), 4.28 (br s, 4H),
3.78 (m, 4H); 13 C (100 MHz, DMSO-d6) δ: 159.4, 157.5, 152.5, 149.9, 139.0, 131.8, 128.9, 119.4,
118.6, 117.6, 117.2, 66.1, 45.5.
3.2.15. 4-(2-(3-Chlorophenyl)-9H-purin-6-yl)morpholine 3o
Method A: Compound 2b (0.14 g, 0.72 mmol) in EtOH, aldehyde 4d (1.1 eq.), andEt3 N (10 eq.) was kept at 40 ◦ C for 10 days. The product 3o was isolated as an off-whitesolid (0.10 g, 0.32 mmol, 44%); m.p. = 265–267 ◦ C (Found: C, 56.21; H, 4.26; N, 21.66.
C15 H14 ClN5 O.0.3H2 O requires C, 56.10; H, 4.55; N, 21.81); IR (Nujol mull) νmax: 3103,
1587, 1571 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.16 (s, 1H, NH), 8.18 (s, 1H), 8.55–7.48 (m,2H), 8.32–8.28 (m, 2H), 4.29 (br s, 4H), 3.76 (t, J = 4.8 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ:
155.2, 152.9, 152.7, 140.7, 139.4, 133.1, 130.2, 129.3, 127.0, 126.1, 118.3, 66.2, 45.1.
3.2.16. 3-(6-Morpholino-9H-purin-2-yl)phenol 3p
Method B: Compound 2a (0.31 g, 1.58 mmol) in EtOH, aldehyde 4e (1.1 eq.), and TFA(1.5 eq.) was kept at 22 ◦ C for 7 days. Then, the reaction continued in DMSO and Et3 N(10 eq.) at 50 ◦ C for 1 month. The product 3p was isolated as an off-white solid (0.24 g, 0.81
mmol, 51%); (Found: C, 60.53; H, 5.02; N, 23.52. C15 H15 N5 O2 requires C, 60.60; H, 5.09; N,
23.56); 1 H (300 MHz, DMSO-d6) δ: 13.07 (br s, 1H), 9.45 (br s, 1H), 8.13 (s, 1H), 7.77–7.79(m, 2H), 7.23 (t, J = 7.8 Hz, 1H), 6.82 (ddd, J = 7.8 Hz, J = 3.3 Hz, J = 0.9 Hz, 1H), 4.28 (br s,
4H), 3.76 (m, 4H); 13 C (75 MHz, DMSO-d6 δ: 157.3, 156.9, 152.9, 152.6, 139.9, 138.8, 129.2,
118.5, 117.9, 116.6, 114.4, 66.3, 45.1.
3.2.17. 4-(2-(4-Chlorophenyl)-9H-purin-6-yl)morpholine 3q
Method A: Compound 2b (0.31 g, 1.58 mmol) in EtOH, aldehyde 4f (1.1 eq.), and Et3 N(10 eq.) was kept at 20 ◦ C for 13 days and then at 50 ◦ C for 21 h. The product 3q was
isolated as an off-white solid (0.15 g, 0.47 mmol, 30%); m.p. = 284 ◦ C (dec.) (Found: C,
57.00; H, 4.51; N, 22.05. C15 H14 ClN5 O requires C, 57.06; H, 4.47; N, 22.18); IR (Nujol mull)νmax: 1583 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.13 (s, 1H), 8.34 (s, 1H), 8.16 (d, J = 9.0 Hz,2H), 7.50 (d, J = 9.0 Hz, 2H), 4.28 (br s, 4H), 3.75 (m, 4H); 13 C (75 MHz, DMSO-d6) δ: 155.8,
153.0, 152.5, 139.0, 137.3, 134.4, 129.3, 128.3, 118.1, 66.2, 45.3.

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12 of 173.2.18. 4-(2-(3,4-Dichlorophenyl)-9H-purin-6-yl)morpholine 3r
Method A: Compound 2b (0.31 g, 1.57 mmol) in EtOH, aldehyde 4p (1.1 eq.), andEt3 N (10 eq.) was kept at 40 ◦ C for 2 days and then at 50 ◦ C for 6 days. The product 3t was
isolated as an off-white solid (0.18 g, 0.51 mmol, 33%); m.p. > 300 ◦ C (Found: C, 51.28; H,
3.91; N, 19.93. C15 H13 Cl2 N5 O requires C, 51.45; H, 3.74; N, 20.00); IR (Nujol mull) νmax:
3010, 1606, 1584, 1561 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.05 (br s, 1H), 8.45 (d, J = 2.1 Hz,
1H), 8.29 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 8.20 (s, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.29 (br s, 4H),
3.76 (t, J = 4.8 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ: 154.5, 152.9, 139.3, 139.1, 132.2, 131.2,
130.6, 128.9, 127.5, 118.2, 66.2, 45.2.
3.2.19. 4-(2-(4-Chloro-3-(trifluoromethyl)phenyl)-9H-purin-6-yl)morpholine 3s
Method A: Compound 2b (0.27 g, 1.38 mmol) in EtOH, aldehyde 4o (1.1 eq.), andEt3 N (10 eq.) was kept at 22 ◦ C for 7 days and then at 40 ◦ C for 5 days. The product 3u
was isolated as an off-white solid (0.28 g, 0.76 mmol, 55%); m.p. = 248–250 ◦ C (Found: C,
50.00; H, 3.53; N, 18.28. C16 H13 ClF3 N5 O requires C, 50.00; H, 3.39; N, 18.25); IR (Nujol
mull) νmax: 3191, 1587, 1575 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.24 (s, 1H, NH), 8.72 (d,J = 2.1 Hz, 1H), 8.60 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 8.21 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H),
4.30 (br s, 4H), 3.76 (t, J = 4.5 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ: 156.3, 152.9, 152.4,
139.3, 137.9, 132.6, 131.8, 131.6 (q, J = 1Hz), 126.6 (q, J = 1Hz), 126.6 (q, J = 31 Hz), 126.0 (q,J = 6 Hz), 122.9 (q, J = 271 Hz), 118.3, 66.2, 45.1.
3.2.20. 4-(2-(2,5-Dichlorophenyl)-9H-purin-6-yl)morpholine 3t
Method A: Compound 2b (0.28 g, 1.45 mmol) in EtOH, aldehyde 4h (1.1 eq.), andEt3 N (10 eq.) was kept at 22 ◦ C for 6 days and then at 40 ◦ C for 6 days. The product 3r was
isolated as an off-white solid (0.21 g, 0.60 mmol, 41%); m.p. = 259–261 ◦ C (Found: C, 51.26;
H, 3.69; N, 19.98. C15 H13 Cl2 N5 O requires C, 51.45; H, 3.74; N, 20.00); IR (Nujol mull) νmax:
3090, 1602, 1586, 1561 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.23 (br s, 1H, NH), 8.22 (s, 1H),
7.76 (d, J = 2.1 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.50 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 4.24 (brs, 4H), 3.72 (t, J = 4.5 Hz, 4H); 13 C (75 MHz, DMSO-d6) δ: 156.3, 152.7, 152.0, 140.2, 139.3,
131.9, 131.4, 131.0, 130.4, 129.6, 117.7, 66.2, 45.2.
3.2.21. 5-Chloro-2-(6-morpholino-9H-purin-2-yl)phenol 3u
Method B: Compound 2b (0.18 g, 0.91 mmol) in EtOH, aldehyde 4k (1.1 eq.), and TFA(1.0 eq.) was kept at 22 ◦ C for 2 days. Then, the reaction continued in EtOH and Et3 N(10 eq.) at 40 ◦ C for 24 days. The product 3s was isolated as an off-white solid (0.08 g,
0.24 mmol, 26%); m.p. > 300 ◦ C. (Found: C, 54.33; H, 4.23; N, 21.31. C15 H14 ClN5 O2 requires
C, 54.31; H, 4.25; N, 21.11); 1 H (400 MHz, DMSO-d6) δ: 13.76 (s, 1H), 13.32 (br s, 1H), 8.27
(d, J = 2.8 Hz, 1H), 8.23 (s, 1H), 7.34 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H),
4.27 (br s, 4H), 3.78 (t, J = 4.8 Hz, 4H); 13 C (100 MHz, DMSO-d6) δ: 158.2, 156.2, 152.5, 149.6,
139.3, 131.4, 127.7, 122.2, 120.7, 119.2, 117.9, 66.1, 45.4.
3.2.22. 4-(2-(2-Fluoro-5-methoxyphenyl)-9H-purin-6-yl)morpholine 3v
Method A: Compound 2b (0.35 g, 1.80 mmol) in EtOH, aldehyde 4l (1.1 eq.), and Et3 N(10 eq.) was kept at 20 ◦ C for 19 days and then at 40 ◦ C for 7 days. The product 3v was
isolated as an off-white solid (0.17 g, 0.52 mmol, 29%); m.p. = 261–263 ◦ C (Found: C, 57.99;
H, 4.89; N, 20.98. C16 H16 FN5 O2 requires C, 58.35; H, 4.90; N, 21.27); IR (Nujol mull) νmax:
1579, 1500 cm−1 ; 1 H (300 MHz, DMSO-d6) δ: 13.17 (s, 1H), 8.18 (s, 1H), 7.50 (dd, J = 6.6 Hz,
J = 3.6 Hz, 1H), 7.17 (dd, J = 10.5 Hz, J = 9.0 Hz, 1H), 7.02 (dt, J = 3.6 Hz, J = 3.3 Hz, 1H),
4.24 (br s, 4H), 3.77 (s, 3H), 3.72 (m, 4H); 13 C (75 MHz, DMSO-d6) δ: 154.7 (d, J = 244 Hz),155.2 (d, J = 5 Hz), 155.0 (d, J = 2 Hz), 152.9, 152.3, 139.2, 127.8 (d, J = 12 Hz), 117.7, 117.4 (d,J = 25 Hz), 116.0 (d, J = 8 Hz), 115.7 (d, J = 2 Hz), 66.2, 55.6, 45.1.

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13 of 173.2.23. 4-(2-(2,5-Difluorophenyl)-9H-purin-6-yl)morpholine 3x
Method A: Compound 2b (0.31 g, 1.59 mmol) in EtOH, aldehyde 4m (1.1 eq.), andEt3 N (10 eq.) was kept at 35 ◦ C for 1 month. The product 3x was isolated as an off-whitesolid (0.28 g, 0.88 mmol, 56%); m.p. = 260–262 ◦ C (Found: C, 56.78; H, 4.00; N, 21.89.
C15 H13 F2 N5 O requires C, 56.78; H, 4.13; N, 22.07); IR (Nujol mull) νmax: 1581, 1500 cm−1 ;
1 H (300 MHz, DMSO-d6) δ: 13.22 (s, 1H), 8.19 (s, 1H), 7.75–7.80 (m, 1H), 7.29–7.33 (m, 2H),
4.24 (br s, 4H), 3.73 (br s, 4H); 13 C (75 MHz, DMSO-d6) δ: 157.8 (dd, J = 240 Hz, J = 4 Hz),156.6 (dd, J = 250 Hz, J = 4 Hz), 154.2 (d, J = 3 Hz), 152.9, 152.2, 139.3, 128.7 (dd, J = 11 Hz,
J = 8 Hz), 118.4 (dd, J = 25 Hz, J = 9 Hz), 117.9, 117.4 (dd, J = 23 Hz, J = 11 Hz), 117.3 (dd,J = 23 Hz, J = 5 Hz), 66.2, 45.2.
3.3. Radioligand Binding Assays
The %inhib of specific radioligand binding at the receptors by the compounds wastested at the concentration of 10 µM at all adenosine receptors following the conditions
described below. Competition binding curves at several receptors were made by testing
six different concentrations (ranging from 10 nM to 100 µM) for all of the compounds
showing an %inhib ≥ 80%.
3.3.1. Human A1 Receptor
Competition binding experiments of adenosine A1 receptor were made with membranes from CHO-A1 cells (Euroscreen, Brussels, Belgium). Membranes were defrosted
and suspended in an incubation buffer with 10 mM of MgCl2 , 100 mM of NaCl, 20 mM ofHepes, 2 UI/mL of adenosine deaminase, and pH = 7.4. Each well of a GF/C multiscreen
plate (Millipore, Madrid, Spain), prepared in duplicate, contained 15 µg of protein, 2 nM of[3 H]DPCPX, and the tested compound. Non-specific binding was calculated with 10 µM
of (R)-PIA. The reaction mixture was incubated at room temperature (25 ◦ C) for 60 min,after which samples were filtered and measured in a microplate beta scintillation counter(Microbeta Trilux, Perkin Elmer, Madrid, Spain).
3.3.2. Human A2A Receptor
Competition binding experiments of adenosine A2A receptor were made in membranes
from HeLa-A2A cells, which were provided by Dr. Mengod (Departament de Neuroquimica,
Institut d’Investi-gacions Biomediques de Barcelona, CSIC-IDIBAPS, Barcelona, Spain).
Membranes were defrosted and suspended in an incubation buffer with 1 mM of EDTA, 50
mM of Tris-HCl, 10 mM of MgCl2 , and 2 UI/mL of adenosine deaminase, with pH = 7.4.
Each well of a GF/C multiscreen plate (Millipore, Madrid, Spain), prepared in duplicate,contained 3 nM of [3 H]ZM241385, 10 µg of protein, and the tested compound. Nonspecificbinding was calculated with 50 µM of NECA. The reaction mixture was incubated at room
temperature (25 ◦ C) for 30 min, after which samples were filtered and measured in a
microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).
3.3.3. Human A2B Receptor
Competition binding experiments of adenosine A2B receptor were made in membranes
from HEK-293-A2B cells (Euroscreen, Brussels, Belgium). Membranes were defrosted
and suspended in incubation buffer with 1 mM of EDTA, 10 mM of MgCl2 , 0.1 mM ofbenzamidine, 50 mM of Tris-HCl, 10 µg/mL of bacitracine, and 2 UI/mL of adenosine
deaminase, with pH = 6.5. Each reaction well, prepared in duplicate, contained 35 nMof [3 H]DPCPX, 18 µg of protein, and the tested compound. Non-specific binding was
calculated with 400 µM of NECA. The reaction mixture was incubated at room temperature(25 ◦ C) for 30 min, after which samples were filtered through a multiscreen GF/C microplateand measured in a microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer,
Madrid, Spain).

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14 of 173.3.4. Human A3 Receptor
Competition binding experiments of adenosine A3 receptor were made in membranes
from HeLa-A3 cells, which were provided by Dr. Mengod (Departament de Neuroquimica,
Institut d’Investi-gacions Biomediques de Barcelona, CSIC-IDIBAPS, Barcelona, Spain).
Membranes were defrosted and suspended in incubation buffer with 1 mM of EDTA, 5 mMof MgCl2 , 50 mM of Tris-HCl, and 2 UI/mL of adenosine deaminase, with pH = 7.4. Eachreaction well of a GF/B multiscreen plate (Millipore, Madrid, Spain), prepared in triplicate,contained 90 µg of protein, 30 nM of [3 H]NECA, and the tested compound. Non-specificbinding was determined with 100 µM of (R)-PIA. The reaction mixture was incubated atroom temperature (25 ◦ C) for 180 min, after which samples were filtered and measured in a
microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).
3.4. Functional Studies
3.4.1. Human A1 Receptor
The agonist/antagonist behavior of tested compounds at A1 receptor was determinedin CHO-A1 cells by measuring reversion induced by the tested compound of 10 µM
of NECA-mediated inhibition of forkolin-stimulated cAMP production. Cells grown in
96-well plates with growth medium containing dialyzed fetal bovine serum were washed
2 times with F-12 nutrient mixture medium containing 25 mM of HEPES and 20 µM of thephosphodiesterase inhibitor rolipram, with pH = 7.4. After this time, the tested compounds
were preincubated for 15 min in test medium at different concentrations (ranging from
0.1 nM to 10 µM). After this time, 10 µM of NECA and 3 µM of forskolin were added toeach well. The incubation was continued for 15 min, and the reaction was stopped with alysis buffer from the cAMP enzyme immunoassay kit (GE Healthcare, Chicago, IL, USA).
Cell lysates were put in a plate with anti-IgG and anti-cAMP in the presence of cAMP–
peroxidase during 60 min. Then, the wells were washed 4 times with the wash buffer fromthe kit, and TMB was added to each well and incubated for 60 min. The peroxidase reaction
was stopped with 1 M of sulphuric acid, and the cAMP amount was determined from theoptical density at 450 nm (Tecan M100 reader).
3.4.2. Human A3 Receptor
The agonist/antagonist behavior of tested compounds at A3 receptor was determinedin CHO–A3 cells, which were in-house generated at BioFarma Research Group (Departmentof Pharma-cology, University of Santiago de Compostela, Spain), by measuring reversion
induced by the tested compound of 10 µM of NECA inhibition of forskolin-stimulatedcAMP production. Cells grown in 96-well plates with growth medium containing dialyzed
fetal bovine serum were washed 2 times with DMEM F-12 nutrient medium containing
25 mM of HEPES, 30 µM of rolipram, with pH = 7.4. After this time, the tested compounds
were preincubated for 15 min in test medium at different concentrations (ranging from
0.1 nM to 10 µM). Finally, 10 µM of NECA and 10 µM of forskolin were added to each well.
The incubation lasted 15 min, and cAMP was determined through an enzyme immunoassay(Perkin Elmer) for Human A1 receptor.
3.5. Data Analysis
Concentration response curves were fitted with Prism 10.1.2 (Graph Pad, San Diego,
CA, USA). Radioligand binding pKi values were extrapolated from the equation
pKi = −log(IC50 /(1 + ([L]/KD )))where IC50 is the concentration that displaced specific radioligand binding at 50%, extrapolated from non-linear fitting, [L] is the concentration of radioligand employed, and KD isthe dissociation constant of the radioligand. In cAMP studies, KB values were derived by

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employing the derivation of the Cheng-Prusoff equation, reported by Leffand Dougall in1993 [37].
KB = IC50 /(2 + (A/(EC50 )n )1/n − 1);
where A is the concentration of the agonist employed to stimulate the receptor, EC50 is thepotency of the agonist for stimulating the receptor, and n is the Hill slope of the antagonist’s
concentration-response curve.
3.6. Docking Studies
The molecular docking calculations were carried out using the Molecular Operating
Environment (MOE) software (version 2022.02, https://www.chemcomp.com, accessedon 25 April 2024) and the crystal structure of the A1 receptor in complex with the ligand
DU1 (PDB accession code: 5UEN) [35]. The receptor was prepared by removing nonprotein atoms and adding missing side chains. Engineered mutations were reverted backto the wild-type sequence based on the side chain rotamers determined through MOE. The
protonation states of ionizable residues were set to the most probable at physiological pH.
The binding site was defined based on the co-crystallized ligand, and default parameters in
MOE were used in the docking calculations.
4. Conclusions
In summary, several 6-morpholino-purine derivatives with different substituents in
C-2 and a methyl or a hydrogen at N-9 of the purine ring were synthesized and pharmacologically tested at human A1 , A2A , A2B , and A3 adenosine receptor subtypes. Four of
the twenty-three synthesized purines showed high affinity and selectivity for A1 receptors,
and five showed high affinity and selectivity for A3 receptors. Three compounds (3k, 3q,
and 3u) showed a dual affinity for A1 /A2A , A1 /A3 , and A1 /A2B receptors, respectively. In
addition, three compounds showed high affinity without selectivity for any specific adenosine receptor subtype (3n, 3o, and 3x). SAR studies showed that the substituent groupsin C-2, C-6, and N-9 positions affect the potency and selectivity of the compounds foradenosine receptors. To generate highly potent antagonist ligands for adenosine receptors,
a morpholine group and a hydrogen atom must be present in C-6 and N-9, respectively.
Indeed, 9H-purines bind some 5–358 times more strongly than their corresponding 9-CH3 purines for the adenosine receptors. The substituted aryl group in C-2 is very importantfor governing the selectivity towards the adenosine receptor subtypes. Additionally, ourresults suggested that the A3 receptor subtype presents a larger hydrophobic pocket thanthat of the A1 receptor subtype, as A3 accommodates bigger substituent groups Y present
at the meta position of substituent R1 . In conclusion, several purine-based compounds
are described herein, some with high affinity and antagonist selectivity for A1 , A3 , dual
A1 /A2A , A1 /A2B , and A1 /A3 receptors.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29112543/s1, 1 H and 13 C NMR spectra of representativesynthesized compounds.
Author Contributions: Conceptualization, F.A., M.F.P. and M.A.C.; formal analysis, M.C., J.B., J.C.
and M.A.C.; funding acquisition, J.C., M.I.L., M.F.P. and M.A.C.; investigation, F.A., C.C., A.R., S.T.,
M.C., J.B. and H.H.; methodology, M.C. and M.A.C.; project administration, M.I.L., M.F.P. and M.A.C.;
supervision, J.C., M.F.P. and M.A.C.; writing—original draft, F.A.; writing—review and editing,
M.A.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Portuguese Fundação para a Ciência e Tecnologia/Ministério
da Educação e Ciência (MEC) through funds in the framework of the Strategic Funding of CQUM
(UID/QUI/00686/2020), (UID/QUI/00686/2018), FEDER funds through the Operational Programme
for Competitiveness Factors (COMPETE 2020), Programa Operacional de Competitividade e Internacionalização (POCI) (POCI-01-0145-FEDER-031354) Rede Nacional de RMN (PINFRA/22161/2016), and
PhD grants to Carla Correia and Ashly Rocha (SFRH/BD/22270/2005; SFRH/BD/85937/2012), Xunta
de Galicia (ED431C 2022/20), and the European Regional Development Fund (ERDF). Filipe Areias

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gratefully acknowledges the Post-PhD grant from the Portuguese FCT (SFRH/BPD/26106/2005). J.C.
received funding from the Olle Engkvist Foundation (219-0154) and the Swedish Research Council(2021-4186). The LECO instrument for elemental analysis TruSpec Micro CHN was purchased within
the PT-OPENSCREEN project (NORTE-01—0145-FEDER-085468), financed by CCDR-N.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Conflicts of Interest: The authors declare no conflicts of interest.

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