Design and synthesis of 2,6-disubstituted-4′-selenoadenosine- 5′-N,N-dimethyluronamide derivatives as human A3 adenosine receptor antagonists

Article abstract of DOI:10.3390/ph14040363

A new series of 4′-selenoadenosine-5′-N,N-dimethyluronamide derivatives as highly potent and selective human A3 adenosine receptor (hA3AR) antagonists, is described. The highly selective A3AR agonists, 4′-selenoadenosine-5′-N-methyluronamides were successfully converted into selective antagonists by adding a second N-methyl group to the 5′-uronamide position. All the syn-thesized compounds showed medium to high binding affinity at the hA3AR. Among the synthe-sized compounds, 2-H-N⁶-3-iodobenzylamine derivative 9f exhibited the highest binding affinity at hA3AR. (Ki = 22.7 nM). The 2-H analogues generally showed better binding affinity than the 2-Cl analogues. The cAMP functional assay with 2-Cl-N⁶-3-iodobenzylamine derivative 9l demonstrated hA3AR antagonist activity. A molecular modelling study suggests an important role of the hydrogen of 5′-uronamide as an essential hydrogen bonding donor for hA3AR activation.

Full text of DOI:10.3390/ph14040363

pharmaceuticals
Article
0
Design and Synthesis of 2,6-Disubstituted-4 -Selenoadenosine-
0
5
-N,N-Dimethyluronamide Derivatives as Human A3
Adenosine Receptor Antagonists
Hongseok Choi , Kenneth A. Jacobson 2 , Jinha Yu 1,3,
1
*
and Lak Shin Jeong 1,*
1
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,
Seoul 08826, Korea; choihs906@snu.ac.kr
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892, USA;
kennethj@niddk.nih.gov
2
3
Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea
Correspondence: yuj@kist.re.kr (J.Y.); lakjeong@snu.ac.kr (L.S.J.); Tel.: +82-2-880-7850 (L.S.J.)
*
0
0
Abstract: A new series of 4 -selenoadenosine-5 -N,N-dimethyluronamide derivatives as highly
potent and selective human A adenosine receptor (hA AR) antagonists, is described. The highly
3
3
0
0
selective A AR agonists, 4 -selenoadenosine-5 -N-methyluronamides were successfully converted
into selective antagonists by adding a second N-methyl group to the 5 -uronamide position. All
3
0
the synthesized compounds showed medium to high binding affinity at the hA AR. Among the
synthesized compounds, 2-H-N -3-iodobenzylamine derivative 9f exhibited the highest binding
3
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
6
ꢀ
ꢁꢂꢃꢄꢅꢆ
affinity at hA AR. (K = 22.7 nM). The 2-H analogues generally showed better binding affinity
than the 2-Cl analogues. The cAMP functional assay with 2-Cl-N -3-iodobenzylamine derivative 9l
3
i
Citation: Choi, H.; Jacobson, K.A.;
6
Yu, J.; Jeong, L.S. Design and
demonstrated hA AR antagonist activity. A molecular modelling study suggests an important role
Synthesis of
3
0
0
of the hydrogen of 5 -uronamide as an essential hydrogen bonding donor for hA AR activation.
2
,6-Disubstituted-4 -Selenoadenosine-
3
0
5
-N,N-Dimethyluronamide
0
Derivatives as Human A3 Adenosine
Keywords: A3 adenosine receptor; structure-activity relationship; 4 -Selenonucleosides; antagonist
Receptor Antagonists. Pharmaceuticals
2
021, 14, 363. https://doi.org/
1
0.3390/ph14040363
1
. Introduction
Adenosine, which is the endogenous ligand of the adenosine receptors (ARs), is an
important neuromodulator and mediates through activation of its four receptors, consisting
of A , A , A , and A subtypes. These receptors are widely distributed in tissues and
Academic Editors: Catia Lambertucci
and Rosaria Volpini
1
2A
2B
3
Received: 23 March 2021
Accepted: 9 April 2021
Published: 14 April 2021
involved in various physiological activities [1]. Each subtype couples to a preferred type of
G protein; A and A ARs primarily couple to the G proteins, and A2A and A ARs couple
1
3
i/o
2B
to G
and their physiological roles have been extensively studied [
is an important receptor to regulate cardioprotection in cardiac ischemia [
of neutrophils [ ], and cell proliferation [ ]. These results led to the development of A AR
agonists as anticancer agents [ ]. Selective A AR antagonists are also promising ligands to
modulate inflammation [ ] and cerebroprotection [
antagonists could enhance cancer treatment via the inhibition of HIF-1
s
proteins. A2B and A ARs are also known to be coupled to G
q
proteins. AR signaling
3
2,3]. Among them, the A AR
3
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
4], degranulation
5
6
3
7
3
8
9,10]. Some studies showed that A AR
3
α
and VEGF protein
accumulation in hypoxia and in tumors [11] and are potential anti-glaucoma therapeutics
as they reduce intraocular pressure in mouse and monkey [12].
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
In the past decades, a variety of approaches have been followed to discover novel drug
6
0
candidates targeting A AR. 2-Chloro-N -(3-iodobenzyl)adenosine-5 -N-methyluronamide
3
0
(
Cl-IB-MECA, 1) and its 4 -thio analogue 2 were discovered as potent and selective A AR
3
agonists from the extensive structure-activity relationships based on the structure of adeno-
0
sine [13] (Figure 1). The 5 -uronamide hydrogen of
1
, required for full agonism, forms a
AR as modeled, suggesting that this interac-
tion is essential for receptor activation by adenosine agonists [14]. Consistent with these
putative hydrogen bond with T94 (3.36) at hA
3
4
.0/).
Pharmaceuticals 2021, 14, 363. https://doi.org/10.3390/ph14040363
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 363
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0
findings, the 4 -truncated analogues, such as
3
and
4, lacking this hydrogen bond donor
were discovered to act as A AR antagonists or low-efficacy agonists, demonstrating that
3
0
a hydrogen bond donating ability of the 5 -uronamide promotes A AR activation [15
,
16].
3
The removal of the hydrogen bond donor ability by appending another methyl group
0
0
to the 5 -uronamide, e.g., 5 -N,N-dimethyluronamide derivatives
5 and 6, similarly re-
duced A AR efficacy. These compounds were characterized as potent and selective A AR
3
3
antagonists [17,18].
Figure 1. Conversion of A AR agonists to A AR antagonists.
3
3
0
On the basis of a bioisosteric rationale, we recently reported that 4 -seleno analogues of
1
and
2
, i.e.,
7
and
8
, were discovered as potent and selective hA AR agonists [19
]
(Figure 2).
3
0
0
They exhibited comparable A AR binding affinity as the corresponding 4 -oxo- and 5 -thio
3
nucleosides 1 and 2. However, X-ray analysis indicated that in the pure crystalline state
they preferred a syn nucleobase orientation and a South sugar conformation, unlike
1 and
0
2. As mentioned above, removal of the amide hydrogen of the 5 -uronamide of
1
and
2
by N-methylation, resulting in
5
and
6
successfully converted A AR agonists into A AR
3
3
0
antagonists. Based on these findings, we hypothesized that the 4 -seleno analogue of
5
or
0
6
, bearing a 5 -N,N-dimethyluronamide moiety might be an A AR antagonist (Figure 2).
3
Thus, we analyzed the structure-activity relationship as A AR ligands of this series by
3
6
0
modifying N - and C2 positions, by synthesizing novel 4 -selenonucleosides 9a–l. Herein,
we report the synthesis and biological evaluation of 2,6-disubstituted-4 -selenoadenosine-
-N,N-dimethyluronamide derivatives 9a–l as potent and selective A AR a3ntagonists.
0
0
5
3

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Figure 2. The rationale for the design of the target nucleosides 9a–l.
2. Results
2
.1. Chemistry
0
For the synthesis of final compounds 9a–l, key intermediates, 4 -seleno purine nu-
cleosides 15a–b were synthesized from D-ribose following the previously reported proce-
dures [18 19] (Scheme 1). Briefly, D-ribose was converted to L-lyxonolactone derivative 10
,
in three steps (oxidation to lactone, conversion of D-ribo configuration to L-lyxo config-
uration, and tert-butyldiphenylsililyl (TBDPS) protection). Reduction of 10 with NaBH4,
ring cyclization of resulting diol with selenide ion, and a Pummerer rearrangement of
4
6
-seleno sugar afforded the glycosyl donor 11. A Vorbrüggen condensation of 11 with
9
-chloropurine and 2,6-dichloropurine produced the N -
β
-anomers 12a and 13a with
7
concomitant formation of their corresponding N -
Conversion of N isomers 12b and 13b to their corresponding N isomers 12a and 13a
β-anomers 12b and 13b, respectively.
7
9
was achieved by using TMSOTf at high temperature. Removal of the TBDPS group of
0
1
2a and 13a yielded the 5 -CH OH derivatives 14a and 14b, respectively. Conversion of
2
0
0
the 5 -hydroxymethyl group of 14a and 14b into a 5 -N,N-dimethyluronamide was suc-
cessfully achieved, but the final deprotection of the acetonide group under strongly acidic
conditions resulted in decomposition, instead of giving the desired final products. Thus,
we exchanged the acetonide protecting group of 14a and 14b with a TBS group in four
steps, giving 15a and 15b, respectively. Firstly, a PNB protecting group was attached to the
0
5
-position of 14a and 14b, followed by acetonide group deprotection with 50% aqueous
TFA to give diols. The diols were then protected with a TBS group using TBSOTf followed
by deprotection of the PNB group with sodium hydroxide in 1,4-dioxane to give 15a and
1
5b, respectively. The final deprotection with sodium hydroxide required mild reaction
conditions (room temperature, overnight) because of the possible hydrolytic conversion of
-chloropurine to hypoxanthine.
6

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Scheme 1. Synthesis of intermediates 15a and 15b from D-ribose (a) i. Br , H O, K CO ; ii. Acetone, H SO ; (b) i. MsCl,
2
2
2
3
2
4
◦
Et N, CH Cl , 0 C, 2 h; ii. KOH, H O, rt, 15 h; (c) TBDPSCl, Et N, DMAP, CH Cl , rt, 4 h; (d) NaBH , MeOH, rt, 1 h; (e) i.
MsCl, Et N, CH Cl , rt, 1 h; ii. Se, NaBH , EtOH, THF, 60 C, 15 h; (f) i. mCPBA, CH Cl ,
h; (g) 6-chloropurine, BSA, TMSOTf, PhCH , 95 C, 15 h or 2,6-dichloropurine, BSA, TMSOTf, CH CN, 60 C, 2 h; (h)
TMSOTf, PhCH , 95 C, 30 min; (i) TBAF, THF, rt, 1 h; (j) i. PNBCl, Et N, CH Cl , rt, 1 h; ii. TFA/H O, THF, rt, 2 h; (k) i.
3
2
2
2
3
2
2
4
◦
◦
◦
−
78 C, 1 h; ii. Ac O, 100 C,
3
2
2
4
2
2
2
◦
◦
4
3
3
◦
3
3
2
2
2
TBSOTf, Et N, DMAP, CH Cl , rt, 1 h; ii. NaOH, 1,4-dioxane, rt, 15 h.
3
2
2
Synthesis of the final nucleosides, 9a–l from the key intermediates, 15a and 15b is
shown in Scheme 2. The direct oxidation of the alcohols of 15a and 15b to the carboxylic
acids have been tried with many oxidizing reagents, but none of them could give the
desired acid. Thus, we employed a sequential oxidation method via aldehyde instead of
direct oxidation to the carboxylic acid. Albright–Goldman oxidations of 15a and 15b, using
DMSO as an oxidizing agent under mild condition afforded the aldehydes 16a and 16b
respectively. Tollens oxidation converted the aldehydes 16a and 16b to the corresponding
,
0
carboxylic acids smoothly, which without purification underwent the amide coupling
0
reaction with dimethylamine in the presence of DIPEA, and HATU to yield the 5 -N,N-
dimethyluronamides 17a and 17b, respectively. The TBS deprotection of 17a and 17b with
TBAF and acetic acid gave the diols 18a and 18b, respectively. The key intermediates

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18a and 18b were treated with various amines such as ammonia, alkylamines and 3-
halobenzylamines to yield 2-H derivatives 9a–f and 2-Cl derivatives 9g–l, respectively.
0
0
◦
Scheme 2. Synthesis of 5 -N,N-dimethyluronamide 4 -selenonucleoside analogues 9a–l. (a) DMSO, Ac O, 100 C, 1 h; (b)
2
◦
AgNO , NaOH, NH OH, THF, 0 C, 30 min; (c) (CH ) NH
e) RNH , Et N, EtOH, reflux, 15–30 h.
2 3
·
HCl, DIPEA, HATU, THF, rt, 1 h; (d) TBAF, AcOH, THF, rt, 15 h;
3
4
3 2
(
2
2
.2. Biology
.2.1. Binding Affinity
The binding affinities of all the final compounds 9a–l were evaluated, using radioli-
gand binding assays at four human AR subtypes (Table 1), by reported methods [20]. All of
the final compounds 9a exhibited medium to high binding affinity at the hA AR, while no
binding affinity at other subtypes such as hA , hA , and hA ARs was observed. Among
–
l
3
1
2A
2B
the tested compounds, compound 9f exhibited the highest affinity (K = 22.7 nM) at hA AR,
i
3
0
0
which is comparable to the corresponding 4 -oxo- and 4 -thio analogues 5 (K = 29.0 nM)
(K = 15.5 nM). The introduction of a 3-halobenzyl group at the N position increased
the hA AR binding affinity when compared to the N -unsubstituted adenine compounds
i
6
and
6
i
6
3
9a or 9g, indicating that a favorable hydrophobic interaction exits at the hA AR binding
3
site. In the 2-H series, the binding affinity of 3-halobenzyl derivatives 9c–f was decreased
in the following order: 3-I-benzyl 9f > 3-Br-benzyl 9e > 3-Cl-benzyl 9d > 3-F-benzyl 9c
The halogen size correlated with hA AR binding affinity, whereas in the 2-Cl series, the
.
3
binding affinity of 3-halobenzyl derivatives 9i
–
l
was almost same within the range of
0
0
180–250 nM. In general, the 4 -seleno-5 -N,N-dimethyluronamide derivatives 9a-l exhibited

Pharmaceuticals 2021, 14, 363
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0
0
lower binding affinity than the 4 -seleno-5 -N-methyluronamide derivatives
7
and
8. It is
6
interesting to note that 2-Cl-N -3-iodobenzyl analogue 9l exhibited much weaker binding
affinity than the corresponding 2-H analogue 9f. This tendency was also found in the
0
0
-N-methyluronamide 4 -seleno derivatives 7 and 8.
5
0
0
Table 1. Binding affinities of known A AR ligands and 5 -N,N-dimethyluronamide-4 -selenonucleoside derivatives (9a–l
)
3
at human A , A2A, A2B, and A ARs.
1
3
a,b
Compound
Affinity, K , nM ± SEM
(or % Inhibition at 10 uM)
i
X
Y
R1
R2
hA AR
hA2AAR
>10,000
>10,000
1080 ± 140
1200 ± 70
22% ± 3
3% ± 2
hA2BAR
>10,000
>10,000
ND
hA AR
3
1
5 c
6 c
7 d
8 d
O
S
Cl
Cl
H
3-I-Bn
3-I-Bn
3-I-Bn
3-I-Bn
H
CH3
CH3
H
5870 ± 930
29.0 ± 4.9
15.5 ± 3.1
0.57 ± 0.10
4.20 ± 0.73
3710 ± 600
609 ± 47
2020 ± 170
1190 ± 160
36.3 ± 12.7
22.7 ± 8.9
3250 ± 370
1060 ± 140
238 ± 37
6220 ± 640
480 ± 94
311 ± 47
16% ± 4
9% ± 2
Se
Se
Se
Se
Se
Se
Se
Se
Se
Se
Se
Se
Se
Se
Cl
H
H
ND
9
a
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
17% ± 1
9% ± 4
19% ± 5
15% ± 1
ND
9
b
H
CH3
9
c
H
3-F-Bn
3-Cl-Bn
3-Br-Bn
3-I-Bn
H
13% ± 2
23% ± 5
43% ± 7
36% ± 6
9% ± 1
58% ± 7
34% ± 2
63% ± 5
58% ± 2
68% ± 2
12% ± 1
4% ± 3
37% ± 8
32% ± 1
3% ± 3
5% ± 2
9% ± 4
13% ± 1
10% ± 4
10% ± 1
9
d
H
9
e
H
9
f
H
ND
9
g
Cl
Cl
Cl
Cl
Cl
Cl
25% ± 5
19% ± 5
14% ± 2
27% ± 4
27% ± 6
23% ± 3
9
h
CH3
9
i
j
3-F-Bn
3-Cl-Bn
3-Br-Bn
3-I-Bn
9
195 ± 40
180 ± 12
253 ± 29
9
k
9
l
a
All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR
3
(
[
A
1
AR and A
3
AR in CHO cells, A2AAR in Hela cells and A2BAR in HEK-293 cells). Binding was carried out using 2 nM [ H]DPCPX, 3 nM
ARs, respectively. Values are expressed
SEM (n = 2). When a value is expressed as a percentage, it refers to the percent inhibition of a specific radioligand binding at
3
3
3
H]ZM241385, 25 nM [ H]DPCPX or 0.5 nM [ H]NECA as radioligands for A
1
, A2A, A2B, and A
3
b
as mean
1
±
0 µM, with nonspecific binding defined using 10 µM NECA. Ref [16]. d Ref [19].
c
2.2.2. CAMP Functional Assay
In a cAMP functional assay at hA AR expressed in CHO cells, compound 9l behaved
3
as an antagonist, like compounds
5
and
6
, with K value of 114.5 nM (Figure 3). Like
5
and
B
0
6, an additional methyl group on the 5 -N-methyluronamide converted an agonist into an
antagonist, indicating that amide hydrogen is essential for receptor activation in this series,
0
as well. However, the fact that the 5 -N,N-dimethyluronamide derivatives exhibited weaker
0
binding affinity than the corresponding 5 -N-methyluronamide derivatives demonstrates
that steric effects induced by 5 -N,N-dimethyluronamide reduce the binding affinity at
0
the A AR.
3

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Figure 3. Concentration
inhibition of 10
of duplicate experiments.
−
response curve of 9l in a functional assay at human A AR measuring
3
µ
M NECA-induced cAMP accumulation. Points represent mean
±
SD (vertical bars)
2
.3. Molecular Modelling Studies
0
0
To investigate how 5 -N,N-dimethyluronamide 4 -selenonucleoside derivatives bind
at hA AR, we docked our compounds into the reported homology model of hA AR [21
]
3
3
using Autodock Vina [22]. The most potent compound 9f bound well at the orthosteric
0
0
binding site with a South ring conformation (2 -endo/3 -exo), displaying H-bonds with
0
Ser271 and His272 (Figure 4). Compared to the 5 -N-methyluronamide derivative
5, the
adenine ring still maintained π−π interaction with Phe168 and the iodobenzene ring had
interactions with Val169, Ile253 and Leu264 [19]. The glycosidic bond was in an anti
0
conformation. However, either H-bonding of 5 -N-uronamide with Thr94 or the adenine
with Asn250 was not observed (marked as a red circle in Figure 4), suggesting that this
H-bonding plays a key role in discriminating an agonist from an antagonist.
Figure 4. Predicted binding mode of compound 9f in the homology model of hA AR. Hydrogen
3
bonds are depicted as green dashed line. Hydrophobic interactions are marked with a purple dashed
line and π-π interactions are marked with a pink dashed line.

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3. Materials and Methods
3
.1. Chemical Synthesis
1
13
Proton ( H) and carbon ( C) NMR spectra were obtained on a Jeol JNM-ECA 300
(JEOL Ltd. Tokyo, Japan; 300/75 MHz), Bruker AV 400 (Bruker, Billerica, MA, USA;
4
00/100 MHz), and AMX 500 (Bruker, Billerica, MA, USA; 500/125 MHz) spectrometer.
1
The H NMR data were reported as peak multiplicities: s for singlet; d for doublet; dd for
doublet of doublets; t for triplet; td for triplet of doublet; q for quartet; quin for quintet;
bs for broad singlet and m for multiplet. Coupling constants were reported in hertz.
The chemical shifts were reported as ppm (δ) relative to the solvent peak. All reactions
were routinely carried out under an inert atmosphere of dry nitrogen. IKA RCT basic
type heating mantle was used to provide a constant heat source. Microwave-assisted
reactions were carried out in sealed vessels using a Biotage Initiator + US/JPN (Biotage,
Uppsala, Sweden; part no. 356007) microwave reactor, and the reaction temperatures
were monitored by an external surface IR sensor. High-resolution mass spectra were
measured with electrospray-ionization quadrupole time-of-flight (ESI-Q-TOF) techniques.
Melting points were recorded on a Barnstead electrothermal 9100 instrument and are
uncorrected. Reactions were checked by thin layer chromatography (Kieselgel 60 F254,
Merck, Kenilworth, NJ, US). Spots were detected by viewing under a UV light, and by
colorizing with charring after dipping in a p-anisaldehyde solution. The crude compounds
were purified by column chromatography on a silica gel (Kieselgel 60, 70
−
230 mesh, Merck).
All the anhydrous solvents were redistilled over CaH , or P O , or sodium/benzophenone
2
2
5
prior to the reaction.
2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)tetrahy-
(
droselenophene-2-carbaldehyde (16a). To a solution of 15a [19] (348 mg, 0.60 mmol) in
dimethyl sulfoxide (5 mL) was added acetic anhydride (0.11 mL, 1.2 mmol), and the re-
◦
action mixture was stirred at 100 C for 1 h, cooled to room temperature, and diluted
with dichloromethane (20 mL). The mixture was washed with water (10 mL
×
2), and the
aqueous layer was further extracted with dichloromethane (20 mL 2). The combined
×
organic layers were washed with brine (5 mL), dried (MgSO ), filtered, and concentrated
4
under reduced pressure. The residue was purified by silica gel column chromatography
1
(
hexane–ethyl acetate = 4:1) to give 16a (288 mg, 83%) as a white foam: H NMR (400 MHz,
CDCl3)
δ
9.76 (d, 1 H, J = 2.4 Hz), 8.78 (s, 1 H), 8.32 (s, 1 H), 6.24 (d, 1 H, J = 7.2 Hz), 4.86 (dd,
H, J = 2.6, 7.0 Hz), 4.68 (t, 1 H, J = 3.0 Hz), 4.12 (dd, 1 H, J = 2.4, 2.8 Hz), 0.95 (s, 9 H),
.71 (s, 9 H), 0.12 (s, 6 H), 0.05 (s, 3 H), 194.6,
0.4 (s, 3 H); 13C NMR (100 MHz, CDCl3)
52.3, 152.1, 151.7, 145.3, 132.5, 81.0, 77.4, 74.6, 56.9, 56.8, 53.0, 25.9, 25.7, 18.3, 17.9, 0.21,
4.2, −4.45, −4.55, −5.3.
2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)tetra-
1
0
1
−
−
−
δ
(
hydroselenophene-2-carbaldehyde (16b). Compound 15b [19] (1.49 g, 2.43 mmol) was
converted to 16b (1.22 g, 82%) as a yellow foam, using a procedure similar to that used in
1
the preparation of 16a: H NMR (500 MHz, CDCl )
δ
9.73 (d, J = 2.1 Hz, 1 H), 8.40 (s, 1 H),
4.73 (m, 1 H), 4.63 (t, J = 2.9 Hz, 1 H), 4.11 (s, 1 H), 0.91 (s,
H), 0.71 (s, 9 H), 0.090 (s, 6 H), 0.043 (s, 3 H),
0.38 (s, 3 H); 13C NMR (75 MHz, CDCl3)
194.3, 153.2, 153.0, 152.2, 145.7, 131.3, 81.0, 74.4, 56.7, 52.9, 25.69, 25.66, 25.6, 18.0, 17.6,
4.42, −4.70, −4.81, −4.86, −5.55.
2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(6-chloro-9H-purin-9-yl)-N-meth-
3
6
9
δ
.13 (d, J = 6.8 Hz, 1 H), 4.74
−
−
−
−
(
yltetrahydroselenophene-2-carboxamide (17a). To a solution of AgNO (170 mg, 1.00 mmol)
3
◦
was added 1 N NaOH (1.0 mL, 1.00 mmol) at 0 C. After Ag O was precipitated, 24%
2
ammonia water (0.3 mL) was added to the reaction mixture until the mixture was clear.
−
The solution of 16a (288 mg, 0.50 mmol) in THF (8 mL) was added to the above Tollens’
◦
reagent at 0 C, and the reaction mixture was stirred at the same temperature for 30 min and
diluted with water (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2),
and the aqueous layer was acidified with 1 N HCl solution (3 mL) and extracted with
ethyl acetate (10 mL
×
2). The combined organic layers were washed with brine, dried
(
MgSO ), filtered, and evaporated under reduced pressure to give the crude acid. To a
4

Pharmaceuticals 2021, 14, 363
9 of 14
solution of the crude acid in THF (5 mL) were added 1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5-b]-pyridinium 3-oxide hexafluorophosphate (HATU) (190 mg, 0.50 mmol),
dimethylamine hydrochloride (82 mg, 1.00 mmol), and diisopropylamine (0.19 mL, 1.10 mmol)
at room temperature. The reaction mixture was stirred at same temperature for 1 h and
diluted with ethyl acetate (10 mL). The organic layer was washed with water (5 mL
×
2),
and the aqueous layer was further extracted with ethyl acetate (10 mL 2). The combined
×
organic layers were washed with brine (5 mL), dried (MgSO ), filtered, and concentrated
4
under reduced pressure. The residue was purified by silica gel column chromatography
1
(
hexane–ethyl acetate = 3:1) to give 17a (120 mg, 39%) as a white foam; H NMR (400 MHz,
CDCl3)
δ
8.76 (s, 1 H), 8.67 (bs, 1 H), 6.32 (d, J = 6.8 Hz, 1 H), 4.9 (bs, 1 H), 4.63 (m, 1 H),
.21 (m, 1 H), 3.04 (s, 3 H), 2.99 (s, 3 H), 0.92 (s, 9 H), 0.71 (s, 9 H), 0.08 (d, J = 3.6 Hz, 6 H),
.01 (s, 3 H)
2S,3S,4R,5R)-3,4-Bis((tert-butyldimethylsilyl)oxy)-5-(2,6-dichloro-9H-purin-9-yl)-N-
4
0
(
methyltetrahydroselenophene-2-carboxamide (17b). Compound 16b (623 mg, 1.02 mmol)
was converted to 17b (260 mg, 58%) as a yellow foam, using a procedure similar to that
1
used in the preparation of 17a: H NMR (400 MHz, MeOD)
δ
8.69 (s, 1 H), 6.20 (d, J = 6.4 Hz,
1
0
H), 4.75–4.85 (bs, 1 H), 4.60 (m, 1 H), 4.22 (m, 1 H), 3.02 (s, 3 H), 2.98 (s, 3 H), 0.90 (s, 9 H),
.74 (s, 9 H), 0.06 (s, 6 H), 0.02 (s, 3 H)
2S,3S,4R,5R)-5-(6-Chloro-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetrahydro selenophene
2-carboxamide (18a). To a stirred solution of 17a (80 mg, 0.13 mmol) in THF (3 mL) was
added 1 M tetra-n-butylammonium fluoride in THF solution (0.13 mL, 0.13 mmol) and
acetic acid (7 L, 0.13 mmol), and the reaction mixture was stirred at room temperature
for 15 h. The solvent was evaporated, and the resulting residue was purified by silica gel
(
-
µ
column chromatography (dichloromethane–methanol = 100:1
−
10:1) to give 18a (43 mg,
1
8
5%) as a white solid; H NMR (400 MHz, CDCl )
δ
8.93 (s, 1 H), 8.73 (s, 1 H), 6.37 (d,
3
J = 6.4 Hz, 1 H), 4.95 (dd, J = 6 Hz, 3.2 Hz, 1 H), 4.67 (t, J = 3.8 Hz, 1 H), 4.50 (d, J = 4 Hz,
H), 3.03 (s, 3 H), 2.99 (s, 3 H).
2S,3S,4R,5R)-5-(2,6-Dichloro-9H-purin-9-yl)-3,4-dihydroxy-N-methyltetra hydroselen-
ophene-2-carboxamide (18b). Compound 17b (83 mg, 0.127 mmol) was converted to 18b
1
(
(
1
41 mg, 75%) as a white solid, using a procedure similar to that used in the preparation of
1
8a; H NMR (400 MHz, MeOD)
δ
8.93 (s, 1 H), 6.27 (d, J = 6 Hz, 1 H), 4.88 (m, 1 H), 4.65 (t,
J = 4 Hz, 1 H), 4.50 (d, J = 4.4 Hz, 1 H), 3.04 (s, 3 H), 2.99 (s, 3 H).
General Procedure for the Synthesis of 9a–l
To a stirred solution of 18a−b (1 equiv.) in ethanol were added amine (3 equiv.) and
◦
triethylamine (6 equiv.), and the reaction mixture was stirred at 95 C for 15
−
30 h. All
volatiles were evaporated, and the residue was purified by silica gel column chromatogra-
phy (dichloromethane–methanol = 100:1−10:1) to give 9a-l as a white solid.
(
2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimethyltetra hydroselen-
1
ophene-2-carboxamide (9a). White solid; yield: 54%; H NMR (400 MHz, MeOD)
δ
8.50 (s,
1
(
8
H), 8.19 (s, 1 H), 6.24 (d, J = 5.6 Hz, 1 H), 4.68 (t, J = 4 Hz. 1 H), 4.49 (d, J = 4.8 Hz, 3.03
s, 3 H), 2.98 (s, 3 H); 13C NMR (100 MHz, MeOD)
173.9, 157.5, 154.1, 151.3, 142.0, 120.4,
2.8, 77.7, 56.8, 42.4, 38.3, 36.5; HRMS (FAB) found 373.0537 (calculated for C H N O Se
δ
12
17
6
3
+
(
M + H) 373.0527).
(
2S,3S,4R,5R)-3,4-dihydroxy-N,N-dimethyl-5-(6-(methylamino)-9H-purin-9-yl)tetrahy-
1
droselenophene-2-carboxamide (9b). White solid; yield: 53%; H NMR (400 MHz, MeOD)
δ
1
1
8.43 (s, 1 H), 8.23 (s, 1 H), 6.23 (d, J = 6 Hz, 1 H), 4.67 (t, J = 4 Hz, 1 H), 4.48 (d, J = 4.4 Hz,
H), 3.02 (s, 3 H), 2.97 (s, 3 H); 13C NMR (100 MHz, MeOD)
δ
173.89, 157.0, 154.1, 149.6,
41.3, 120.9, 82.0, 77.6, 62.4, 56.8, 42.4, 38.3, 36.4; HRMS (FAB) found 387.0675 (calculated
+
for C H N O Se (M + H) 387.0684).
13
19
6
3
(
2S,3S,4R,5R)-5-(6-((3-fluorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimeth-
1
yltetrahydroselenophene-2-carboxamide (9c). White solid; yield: 74%; H NMR (400 MHz,
MeOD)
δ 8.46 (s, 1 H) 8.23 (s, 1 H), 7.28 (q, J = 6 Hz, 1 H) 7.15 (d, J = 7.6 Hz, 1 H), 7.07 (d,
J = 9.6 Hz, 1 H), 6.92 (td, J = 8, 2 Hz, 1 H), 6.24 (d, J = 5.6 Hz, 1 H), 4.68 (dd, J = 4.4, 4 Hz,

Pharmaceuticals 2021, 14, 363
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1
1
3
H) 4.48 (d, J = 4.8 Hz, 1 H) 3.02 (s, 3 H), 2.97 (s, 3H); 13C NMR (100 MHz, MeOD)
δ 173.9,
65.8, 163.3, 156.2, 154.2, 150.7, 143.5, 141.6, 131.4, 124.3, 120.8, 115.1, 82.1, 77.6, 56.8, 42.4,
+
8.3, 36.4; HRMS (FAB) found 481.0894 (calculated for C H FN O Se (M + H) 481.0903).
19
22
6
3
(
2S,3S,4R,5R)-5-(6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimeth-
1
yltetrahydroselenophene-2-carboxamide (9d). White solid; yield: 68%; H NMR (400 MHz,
MeOD)
H), 4.68 (dd, J = 4.4 Hz, 4 Hz, 1 H), 4.48 (d, J = 4.8 Hz, 1 H), 3.02 (s, 3 H), 2.97 (s, 3 H);
NMR (100 MHz, MeOD) 173.8, 156.1, 154.2, 143.1, 141.7, 135.5, 131.2, 128.6, 128.3, 128.3,
27.0, 120.8, 82.0, 77.6, 56.9, 42.4, 38.2, 36.4; HRMS (FAB) found 497.0599 (calculated for
δ 8.46 (s, 1 H), 8.24 (s, 1 H), 7.36 (s, 1 H), 7.18–7.28 (m, 4 H), 6.24 (d, J = 5.2 Hz,
1
3
1
C
δ
1
+
C H ClN O Se (M + H) 497.0607).
19
22
6
3
(
2S,3S,4R,5R)-5-(6-((3-bromobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimet-
1
hyltetrahydroselenophene-2-carboxamide (9e). White solid; yield: 75%, H NMR (400 MHz,
MeOD + CDCl = 1:3);
δ
8.42 (s, 1 H), 8.29 (s, 1 H), 7.50 (s, 1 H), 7.36 (d, J = 7.6 Hz, 1 H),
3
7
4
.28 (d, J = 7.6 Hz, 1 H), 7.17 (t, J = 8 Hz, 1 H), 6.17 (d, J = 4.8 Hz, 1 H), 4.76 (bs, 2 H),
.73–4.69 (m, 1 H), 4.44 (d, 4.8 Hz, 1 H), 3.02 (s, 3 H), 2.98 (s, 3 H); 1 C NMR (100 MHz,
3
MeOD + CDCl = 1:3) δ 172.1, 155.0, 153.3, 141.2, 140.6, 130.8, 130.7, 130.5, 126.5, 122.9,
3
1
19.8, 81.3, 77.8, 76.6, 55.9, 41.5, 38.0, 36.3; HRMS (FAB) found 541.0106 (calculated for
+
C H BrN O Se (M + H) 541.0102).
19
21
6
3
(
2S,3S,4R,5R)-3,4-dihydroxy-5-(6-((3-iodobenzyl)amino)-9H-purin-9-yl)-N,N-dimeth-
1
yltetrahydroselenophene-2-carboxamide (9f). White solid; yield: 73%, H NMR (400 MHz,
MeOD + CDCl =1:3)
δ
8.45 (s, 1 H), 8.27 (s, 1 H), 7.71 (s, 1 H), 7.56 (d, J = 7.4 Hz, 1 H), 7.33
3
(
4
d, J = 7.7 Hz, 1 H), 7.04 (t, J = 7.8 Hz, 1 H), 6.21 (d, J = 5.4 Hz, 1 H), 4.78–4.76 (m, 1 H),
1
3
.74 (bs, 2 H), 4,71–4.69 (m, 1 H), 4.46 (d, J = 4.9 Hz, 1 H), 3.03 (s, 3 H), 2.99 (s, 3 H);
C
6
NMR (100 MHz, DMSO-d )
δ
173.5, 156.1, 154.3, 142.5, 141.7, 137.8, 137.7, 131.6, 128.1, 120.9,
9
5.6, 82.3, 79.2, 78.8, 77.7, 57.0, 42.5, 38.9, 37.2; HRMS (FAB) found 588.9971 (calculated for
+
C H IN O Se (M + H) 588.9963).
19
21
6
3
(
2S,3S,4R,5R)-5-(2-chloro-6-amino-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimethyltetrah-
1
ydroselenophene-2-carboxamide (9g). White solid; yield: 58%; H NMR (400 MHz, DMSO-
8.33 (s, 1 H), 5.98 (d, J = 4.8 Hz, 1 H), 5.78 (d, J = 4 Hz, 1 H), 5.48 (d, J = 3.2 Hz, 1 H),
.74 (bs, 1 H), 4.50 (bs, 1 H), 4.32 (d, J = 2.8 Hz, 1 H), 2.91 (s, 3 H), 2.87 (s, 3 H); C NMR
6
d )
4
(
δ
1
3
6
100 MHz, DMSO-d )
δ
173.7, 157.5, 154.1, 151.3, 141.9, 120.3, 81.9, 77.5, 49.6, 42.2, 38.1, 36.3;
+
HRMS (FAB) found 407.0132 (calculated for C H ClN O Se (M + H) 407.0138).
12
16
6
3
(
2S,3S,4R,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-dimeth-
1
yltetrahydroselenophene-2-carboxamide (9h). White solid; yield: 69%; H NMR (400 MHz,
DMSO-d )
H), 4.74 (bs, 1 H), 4.50 (bs, 1 H), 4.32 (d, J = 2.8 Hz, 1 H), 2.91 (s, 3 H), 2.87 (s, 3 H);
NMR (100 MHz, DMSO-d )
6
δ
8.33 (s, 1 H), 5.98 (d, J = 4.8 Hz, 1 H), 5.78 (d, J = 4 Hz, 1 H), 5.48 (d, J = 3.2 Hz,
1
3
1
C
6
δ
171.0, 155.5, 153.4, 149.8, 139.6, 118.3, 79.5, 75.4, 64.9, 55.1,
+
4
1.1, 37.1, 35.3; HRMS (FAB) found 421.0293 (calculated for C H ClN O Se (M + H)
13 18 6 3
4
21.0294).
2S,3S,4R,5R)-5-(2-chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-
(
1
dimethyltetrahydroselenophene-2-carboxamide (9i). White solid; yield: 71%; H NMR
400 MHz, MeOD) 8.43 (s, 1 H), 7.29 (m, 1 H), 7.17 (d, J = 6.8 Hz, 1 H), 7.09 (d, J = 10 Hz,
H), 6.94 (m, 1 H), 6.15 (d, J = 5.2 Hz, 1 H), 4.76 (m, 1 H), 4.66 (t, J = 4 Hz, 1 H), 4.47 (d,
J = 5.2 Hz, 1 H); 13C NMR (100 MHz, MeOD)
173.8, 165.7, 163.3, 156.5, 152.0, 143.5, 142.0,
31.4, 129.7, 126.5, 124.6, 115.3, 82.1, 77.6, 57.0, 42.4, 38.3, 36.5; HRMS (FAB) found 515.0518
(
1
δ
δ
1
+
(
calculated for C H ClFN O Se (M + H) 515.0513).
19 21 6 3
(
2S,3S,4R,5R)-5-(2-chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-
1
dimethyltetrahydroselenophene-2-carboxamide (9j). White solid; yield: 69%; H NMR
(
4
5
400 MHz, MeOD)
δ
8.43 (s 1 H), 7.38 (s, 1 H), 7.21–7.30 (m, 3 H), 6.15 (d, J = 5.6 Hz, 1 H),
.76 (dd, J = 5.6 Hz, 3.2 Hz, 1 H), 4.71 (bs, 1 H), 4.65 (dd, J = 4.8 Hz, 3.2 Hz, 1 H), 4.47 (d,
.2 Hz, 1 H), 3.03 (s, 3 H), 2.98 (s, 3 H); 13C NMR (100 MHz, MeOD)
173.8, 156.6, 155.7,
δ
1
52.0, 142.7, 142.0, 135.5, 131.2, 128.9, 128.4, 127.3, 119.3, 82.1, 77.6, 57.0, 44.7, 42.4, 38.3, 36.5;
+
HRMS (FAB) found 531.0211 (calculated for C H Cl N O Se (M + H) 531.0217).
19
21
2
6
3

Pharmaceuticals 2021, 14, 363
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(
2S,3S,4R,5R)-5-(6-((3-bromobenzyl)amino)-2-chloro-9H-purin-9-yl)-3,4-dihydroxy-N,N-
1
dimethyltetrahydroselenophene-2-carboxamide (9k). White solid; yield: 94%; H NMR
(
6
1
400 MHz, MeOD)
δ
8.43 (s, 1 H), 7.54 (s, 1 H), 7.36 (m, 2 H), 7.33–7.39 (t, J = 7.6 Hz, 1 H),
.15 (d, J = 5.6 Hz, 1 H), 4.76 (dd, J = 6.4 Hz, 3.2 Hz, 1 H), 4.71 (bs, 1 H), 4.65 (dd, J = 3.2 Hz,
H), 4.48 (d, J = 4.8 Hz, 1 H), 3.03 (s, 3 H), 2.98 (s, 3 H); 13C NMR (100 MHz, MeOD)
174.5,
δ
1
57.3, 156.5, 152.7, 143.6, 142.7, 132.6, 132.1, 128.4, 124.2, 82.8, 78.3, 57.6, 45.3, 43.0, 38.9, 37.1;
+
HRMS (FAB) found 574.9708 (calculated for C H BrClN O Se (M + H) 574.9712).
19
21
6
3
(
2S,3S,4R,5R)-5-(2-chloro-6-((3-iodobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxy-N,N-
1
dimethyltetrahydroselenophene-2-carboxamide (9l). White solid; yield: 78%; H NMR
(
7
2
400 MHz, MeOD)
δ
8.43 (s, 1 H), 7.75 (s, 1 H), 7.57 (d, J = 4 Hz, 1 H), 7.36 (d, J = 3.6 Hz, 1 H),
.07 (t, J = 8 Hz, 1 H), 6.15 (d, J = 5.6 Hz, 1 H), 4.76 (dd, J = 5.2 Hz, 3.2 Hz, 1 H), 4.64–4.68 (m,
H), 4.48 (d, J = 4.8 Hz, 1 H), 3.03 (s, 3 H), 2.98 (s, 3 H); 13C NMR (100 MHz, MeOD)
173.8,
δ
1
3
56.8, 155.9, 152.0, 142.8, 142.0, 138.0, 137.6, 131.5, 128.3, 95.1, 82.1, 77.6, 57.0, 44.5, 42.4, 38.3,
+
6.5.; HRMS (FAB) found 622.9584 (calculated for C H ClIN O Se (M + H) 622.9574).
19
21
6
3
3.2. Biological Evaluation
3
.2.1. Binding Assay at hA AR
1
Adenosine A receptor competition binding experiments were carried out in mem-
1
branes from CHO-A cells (Euroscreen, Gosselies, Belgium). On the day of assay, mem-
1
branes were defrosted and re-suspended in incubation buffer 20 mM Hepes, 100 mM NaCl,
1
0 mM MgCl , 2 UI/mL adenosine deaminase (pH = 7.4). Each reaction well of a GF/C
2
multiscreen plate (Millipore, Madrid, Spain), prepared in duplicate, contained 15
µ
g of
3
protein, 2 nM [ H]DPCPX and test compound. Nonspecific binding was determined in
◦
the presence of 10
µ
M (R)-PIA. The reaction mixture was incubated at 25 C for 60 min,
after which samples were filtered and measured in a microplate beta scintillation counter
Microbeta Trilux, Perkin Elmer, Madrid, Spain).
(
3.2.2. Binding Assay at hA2AAR
Adenosine A2A receptor competition binding experiments were carried out in mem-
branes from HeLa-A2A cells. On the day of assay, membranes were defrosted and re-
suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl and 2 UI/mL
2
adenosine deaminase (pH = 7.4). Each reaction well of a GF/C multiscreen plate (Millipore,
3
Madrid, Spain), prepared in duplicate, contained 10
µ
g of protein, 3 nM [ H]ZM241385
and test compound. Nonspecific binding was determined in the presence of 50
µ
M NECA.
◦
The reaction mixture was incubated at 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
.2.3. Binding Assay at hA AR
2B
Adenosine A2B receptor competition binding experiments were carried out in mem-
branes from HEK-293-A2B cells (Euroscreen, Gosselies, Belgium) prepared following the
provider’s protocol. On the day of assay, membranes were defrosted and re-suspended
in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl , 0.1 mM benzamidine,
2
1
0
µ
g/mL bacitracine and 2 UI/mL adenosine deaminase (pH = 6.5). Each reaction well
3
prepared in duplicate contained 18
µg of protein, 35 nM [ H]DPCPX and test compound.
Nonspecific binding was determined in the presence of 400
mixture was incubated at 25 C for 30 min, after which samples were filtered through
a multiscreen GF/C microplate and measured in a microplate beta scintillation counter
µ
M NECA. The reaction
◦
(
Microbeta Trilux, Perkin Elmer, Madrid, Spain).
3
.2.4. Binding Assay at hA AR
3
Adenosine A receptor competition binding experiments were carried out in a mul-
3
tiscreen GF/B 96-well plate (Millipore, Madrid, Spain) pretreated with binding buffer
Tris-HCl 50 mM, EDTA 1 mM, MgCl 5 mM, 2 U/mL adenosine deaminase, pH = 7.4).
(
2

Pharmaceuticals 2021, 14, 363
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In each well was incubated 30
in laboratory (Lot: A005/05-07-2019, protein concentration = 3925
µ
g of membranes from Hela-A cell line and prepared
3
3
µg/mL), 10 nM [ H]-
NECA (26.3 Ci/mmol, 1 mCi/mL, Perkin Elmer NET811250UC) and compounds studied
in standard methods. Nonspecific binding was determined in the presence of R-PIA 100
µM
(
Sigma P4532, Sigma-Aldrich, St. Louis, MO, USA). The reaction mixture (Vt: 200
µ
L/well)
◦
was incubated at 25 C for 180 min, after filtered and washed six times with 250
µL wash
buffer (Tris-HCl 50mM pH = 7.4), before measuring in a microplate beta scintillation
counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).
3.2.5. Cyclic AMP Accumulation Assay
Human adenosine A receptor functional experiments were carried out in CHO-A #18
3
3
cell line. The day before the assay, the cells were seeded on the 96-well culture plate
(
(
Falcon 353072, Corning, Glendale, AZ, USA). The cells are washed with wash buffer
Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062), 25 mM
Hepes; pH = 7.4). Wash buffer is replaced by incubation buffer (Dulbecco’s modified eagle’s
medium nutrient mixture F-12 ham (Sigma D8062), 25 mM Hepes, 30 M Rolipram (Sigma
µ
R6520); pH = 7.4). Test compounds and MRS1220 as reference compound (Sigma M228) are
◦
0
added and the cells incubated at 37 C for 15 min. After, 0.1
µ
M of 5 -N-ethylcarboxamido-
◦
adenosine (NECA) (Sigma E2387) is added and the cells incubated at 37 C for 10 min.
Forskolin (Sigma F3917, Sigma-Aldrich, St. Louis, MO, USA) is added and incubated at
◦
3
7 C for 5 min. After incubation, the amount of cAMP is determined using a cAMP Biotrak
Enzymeimmunoassay (EIA) System Kit (GE Healthcare RPN225, GE Healthcare, Chicago,
IL, USA).
3.3. Molecular Modelling
The hA AR homology model was obtained from reference 20. Autodock Vina 4 (The
3
Scripps Research Institute, La Jolla, CA, USA) was used as the docking tool to generate
ligand-protein complex using the following settings: center_x = 7.288, center_y = 8.071,
−
−
center_z = 51.576, size_x = 40, size_y = 40, size_z = 40, energy_range = 4, exhaustiveness = 8.
The ligand–protein complex with the best IFD score were selected and analyzed. The
molecular graphic figures were generated by Biovia Discovery Studio Visualizer software
(
https://3dsbiovia.com/) (accessed on 13 April 2021.).
4
. Conclusions
6
On the basis of potent and selective antagonist
5
and 6 at the human A AR, N -
3
0
0
substituted-5 -N,N-dimethylcarbamoyl-4 -selenonuceloside derivatives (9a–l) were syn-
thesized from D-ribose and evaluated for their binding affinity toward hARs. All final
compounds exhibited medium to high binding affinity toward A AR with high selectiv-
3
ity compared to other subtypes. Among these derivatives, compound 9f was found to
show the highest binding affinity (K = 22.7 nM) at hA AR, comparable to the correspond-
i
5
and
3
0
0
ing 4 -oxo- and 4 -thio analogues
(K = 29.0 nM) and
6
(K = 15.5 nM). As in the case
i
i
0
0
0
of 4 -oxo- and 4 -thio analogues
5
6
, addition of another methyl group to the 5 -N-
methyluronamide converted an A AR agonist into an A AR antagonist, demonstrating
3
3
the importance of amide hydrogen for receptor activation, which was supported by the
molecular modelling study.
We believe that this study helps to define the pharmacophore needed for receptor
activation or inactivation and will aid in the design of selective A AR ligands by medici-
3
nal chemists.
Author Contributions: Conceptualization, L.S.J.; methodology, J.Y.; software, J.Y.; validation, L.S.J.
and J.Y.; formal analysis, H.C.; investigation, H.C. and K.A.J.; data curation, H.C.; writing—original
draft preparation, J.Y.; writing—review and editing, L.S.J.; supervision, L.S.J.; project administration,
L.S.J.; funding acquisition, L.S.J. All authors have read and agreed to the published version of
the manuscript.

Pharmaceuticals 2021, 14, 363
13 of 14
Funding: This research was funded by the National Research Foundation (NRF) of Korea (NRF-
2018R1D1A1A02085459 and 2021R1A2B5B02001544) and the Korea Institute of Science and Technol-
ogy (KIST); ZIADK31117 (NIDDK).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
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