Discovery of 7-(Prolinol-N-yl)-2-phenylamino-thiazolo[5,4-d]pyrimidines as Novel Non-Nucleoside Partial Agonists for the A2A Adenosine Receptor: Prediction from Molecular Modeling

Article abstract of DOI:10.1021/acs.jmedchem.6b00552

We describe the identification of 7-(prolinol-N-yl)-2-phenylamino-thiazolo[5,4-d]pyrimidines as a novel chemotype of non-nucleoside partial agonists for the A_2A adenosine receptor (A_2AAR). Molecular-modeling indicated that the (S)-2-hydroxymethylene-pyrrolidine could mimic the interactions of agonists' ribose, suggesting that this class of compounds could have agonistic properties. This was confirmed by functional assays on the A_2AAR, where their efficacy could be associated with the presence of the 2-hydroxymethylene moiety. Additionally, the best compound displays promising affinity, selectivity profile, and physicochemical properties.

Full text of DOI:10.1021/acs.jmedchem.6b00552

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Brief Article
Discovery of 7-(prolinol-N-yl)-2-phenylamino-thiazolo[5,4-
d]pyrimidines as novel non-nucleoside partial agonists for the
2A
A adenosine receptor: Prediction from molecular modeling
Sandip B. Bharate, Baljinder Singh, Sonja Kachler, Ana Oliveira, Vikas Kumar, Sonali
S. Bharate, Ram A. Vishwakarma, Karl-Norbert Klotz, and Hugo Gutiérrez-de-Terán
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00552 • Publication Date (Web): 26 May 2016
Downloaded from http://pubs.acs.org on May 27, 2016
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Journal of Medicinal Chemistry
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Discovery of 7-(prolinol-N-yl)-2-phenylamino-thiazolo[5,4-
d]pyrimidines as novel non-nucleoside partial agonists for the A2A
adenosine receptor: Prediction from molecular modeling
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Sandip B. Bharate,‡,†,$,*Baljinder Singh,†,⊥,$ Sonja Kachler,║ Ana Oliveira,# Vikas Kumar,×, † Sonali S.
Bharate,× Ram A. Vishwakarma,‡,† Karl-Norbert Klotz,║ and Hugo Gutiérrez de Terán#,*
‡Medicinal Chemistry Division,†Academy of Scientific & Innovative Research (AcSIR), ⊥Natural Products Chemistry
Division, ×Preformulation Laboratory, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India
║Institut für Pharmakologie und Toxikologie, Julius-Maximilians-Universität Würzburg, Versbacher Str. 9, D-97078
Würzburg, Germany
#Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-751 24, Uppsala, Sweden
$ S.B.B. and B.S. contributed equally as a first author.
KEYWORDS. Thiazolo[5,4-d]pyrimidines, adenosine, A2A adenosine receptor agonist, non-nucleoside A2A agonist, GPCR.
ABSTRACT: We describe the identification of 7-(prolinol-N-yl)-2-phenylamino-thiazolo[5,4-d]pyrimidines as a novel chemotype
of non-nucleoside partial-agonists for the A2A adenosine receptor (A2AAR). Molecular-modeling indicated that the (S)-2-
hydroxymethylene-pyrrolidine could mimic the interactions of agonists’ ribose, suggesting that this class of compounds could have
agonistic properties. This was confirmed by functional assays on the A2AAR, where their efficacy could be associated to the
presence of the 2-hydroxymethylene moiety. Additionally, the best compound displays promising affinity, selectivity profile and
physicochemical properties.
benzenepropanoic acid
5 (CGS21680), another moderately
Introduction
A2AAR-selective agonist, displays binding affinities of 27 and 19
Adenosine (1, Figure 1), an endogenous purine nucleoside, is also
the natural ligand for the adenosine receptors (ARs). Four distinct
AR subtypes exist, designated as A1, A2A, A2B, and A3, all
belonging to the G protein-coupled receptor (GPCR) superfamily.
The subtypes A1 and A3 are coupled to Gi which inhibits adenylyl
cyclase and thereby the production of cAMP from ATP, whereas
A2A and A2B are coupled to Gs which activates this enzyme and
nM at the human and rat A2A receptor, respectively.13,
14
Regadenoson (CVT3146, 6), is an adenosine derivative bearing a
N-pyrazole at its 2-position. This compound has been approved by
the FDA as a coronary vasodilator,15,
demonstrating the
16
therapeutic potential of A2A receptor agonists. The only class of
non-nucleoside AR agonist are 2-amino-3,5-dicyanopyridines,17
from which 2-((1H-imidazol-2-yl)methylthio)-6-amino-4-(3-
hydroxyphenyl)pyridine-3,5-dicarbonitrile 7 (LUF5835) exerts
good affinity for A2A receptor.18 The chemical structures of
representative A2A agonists 1-7 are shown in Figure 1.
consequently the cAMP-dependent pathways.1, All adenosine
2
receptor subtypes are characterized by a unique pharmacological
profile.1-7
Adenosine behaves as a high affinity agonist for the A1, A2A,
and A3 receptors (hA1 Ki = 310 nM, hA2A Ki = 700 nM, hA3 Ki =
290 nM) and with considerably lower affinity for the A2B receptor
(hA2B Ki ≥ 10 μM).5 Chemically, adenosine is a nucleoside
comprising a molecule of the purine base adenine attached to a
ribose moiety via a glycosidic bond. The unmodified adenosine
has been of restricted interest in studying adenosine receptors
because it is readily metabolized by a number of enzymes. The
main approach being used to discover AR agonists has been the
NH2
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
OH
OH
Bu
O
O
n
O
HO
HO
HO
H
O
H
N
OH
N
HO
HO
HO
O
O
OH
Adenosine (1)
A2A: Ki = 700 nM
NECA (2)
A2A: Ki = 20 nM
CV1808 (4)
HENECA (3)
A2A: Ki = 6.4 nM
A2B: EC50 = 6100 nM
A1: Ki = 60 nM
A2A: Ki = 100 nM
A1: Ki = 400 nM
A2B: EC50 = 330 nM
A1: Ki = 14 nM
A3: Ki = 6.2 nM
A2B: EC50 = 24000 nM
A1: Ki = 310 nM
A3: Ki = 290 nM
A3: Ki = 2.4 nM
NH2
COOH
modification of adenosine itself, which has led to most of the
NH2
N
N
NH2
reported A2A agonists.5,
Variations of the hydroxymethyl
8-11
N
N
CN
N
N
O
N
N
N
N
portion of the ribosyl unit of adenosine generated N-
ethylcarboxamidoadenosine (NECA, 2), which showed 35-fold
higher potency (hA2A Ki = 20 nM) for A2A than adenosine (1).
However, NECA is a non-selective agonist. Its analog 2-hexynyl-
NECA (HENECA, 3), which additionally introduces
modifications at the C2 position in the adenine core, exhibits high
affinity at A2AAR (hA2A Ki = 6.4 nM) and 10-fold selectivity over
human A1AR.12 2-Phenylaminoadenosine 4 (CV1808) maintains
substitution at the C2 of the adenine but has an intact ribose
structure, and it presents A2A selectivity over the A1 receptor
(rA2A Ki = 100 nM, rA1 Ki = 400 nM). 4-[2-[[6-Amino-9-(N-
ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]
O
N
N
H
N
S
HO
HN
O
NH
CN
HO
H
HO
N
HO
OH
HO
O
LUF5835 (7)
CGS21680 (5)
A2A: Ki = 27 nM
Regadenoson (6)
A2A: Ki = 290 nM
A2B: EC50 = 10000 nM
A1: Ki = 3770 nM
A2A: Ki = 21 nM
A2B: EC50 = 10 nM
A1: Ki = 4.4 nM
A3: Ki = 104 nM
A2B: EC50 = 361000 nM
A1: Ki = 290 nM
A3: Ki = 67 nM
A3: Ki = 10000 nM
Figure 1. Structures of adenosine A2A receptor agonists 1-7.
A2AAR agonists play an important role in the treatment of
cerebral and cardiac ischemia, epilepsy, thrombosis, and arterial
hypertension; however the drug development in this area is
limited by the lack of novel scaffolds. The identification of newer
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A2AAR agonists has been a challenging task, as observed from the
bearing a 2-hydroxymethyl pyrrolidine at R2 (series 4) specifically
recent efforts of Jacobson and Carlsson,19 who performed docking
screens of 6.7 million commercially available molecules against
active-like conformations of the A2AAR to investigate whether
these structures could guide the discovery of agonists. Though
twenty predicted agonists were confirmed to be A2AAR ligands,
none of these effectively activated ARs.
displace a water molecule essential to maintain the bioactive
conformation of the agonists, by consistently mediating an
internal hydrogen-bond between the ribose and the adenine ring.
By occupying this binding area, these compounds show an extra
hydrogen bond interaction between the hydroxyl group of the
ligand with the conserved His7.43 (Figure 3). Alternatively, in
some compounds the preferred docking solution presents a
hydrogen bond to Ser7.42. Our exhaustive docking exploration
shows that these interactions are compatible with the active-like
conformation of the receptor, and resemble the key interactions of
the ribose of typical AR agonists, as revealed by X-ray
crystallography,25, 27, 28 and probed by site-directed mutagenesis
experiments.29 Based on these findings, we hypothesized that
these prolinol-containing compounds (i.e., series 4, compounds
10b, 10d, 10g, 10l) could act as potential adenosine A2A agonists.
1
2
3
4
5
6
7
8
In the search of safe and selective drugs that regulate this
receptor, a general concern is to achieve chemical novelty and
good solubility values. Thiazolo[5,4-d]pyrimidine is a bioisostere
of purine in which the imidazole moiety is replaced by 1,3-
thiazole.20, 21 Previous studies identified thiazolo[5,4-d]pyrimidine
derivatives as antagonists at adenosine A2A receptors.22-24 Using a
computer-aided rational drug design approach, based on the
A2AAR crystal structures, we herein report the discovery and
pharmacological characterization of 7-prolinol linked 2-
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phenylamino-thiazolopyrimidines as
nucleoside A2AAR partial agonists.
a new class of non-
Results and discussion
Design of A2AAR agonists. The design of the series of
compounds presented here started with the consideration of the
thiazolo[5,4-d]pyrimidine as a bioisostere of adenine. Several
substituents were considered to increase binding affinity for the
A2A receptor. The docking studies, in both active-like and inactive
conformations of the A2AAR, indicated that the most common
binding orientation as compared to the adenine core corresponds
to a double flip of the bicyclic scaffold along the two planar axes,
as opposed to the initial bioisosteric design (Figure 2).
NH2
(b)
(a)
Figure 3. Binding mode proposed by molecular docking for compound
10b on the human A2AAR, superimposed with the experimental binding
pose of (a) A2AAR in inactive conformation, in complex with the
antagonist ZM241385 (orange26) and (b) A2AAR in the active-like
conformation bound to the agonist adenosine (magenta25).
NH2
N
N
N
S
N
N
N
N
N
N
N
OH
OH
N
H
N
N
N
H
N
O
Synthesis. Based on the molecular modeling design, a series of
7-substituted thiazolo[5,4-d]pyrimidines 8-10, 13 were
synthesized using the synthetic protocol as depicted in Scheme
1.30 In order to establish structure-activity relationships, various
secondary amines including morpholine, piperidine, diethylamine,
dimethylamine, piperazine, N-alkylpiperazine, pyrrolidine and 2-
hydroxymethylene pyrrolidine (prolinol) were employed for
substitution at C-7 position of the thiazolo[5,4-d]pyrimidine
scaffold. The thiazolo[5,4-d]pyrimidines 13a-l were synthesized
from 4,6-dichloro-5-aminopyrimidine (11) and the corresponding
isothiocyanate (12) using KF/alumina as a catalyst with overall
52-96% isolated yield. The thiazolo[5,4-d]pyrimidine 13i was
prepared by treating 11 with benzoyl isothiocyanate. Compounds
8-10 were synthesized from 2-amino-7-chlorothiazolo[5,4-
d]pyrimidines 13a-h by treating with the corresponding amines 14
with overall yield of 85-98%. For synthesis of prolinol linked
analogs 10b, 10d, 10e, 10g, 10j, 10l and 10n, (S)-2-
hydroxymethylene pyrrolidine (L-prolinol) was used. The
OH
Adenosine
thiazolo[5,4-d]pyrimidine
Adenine
9H-purine
R1
NH2
N
N
R1
S
N
4
3
S
HN
N
N
N
N
N
OH
OH
5
2
HN
N
O
OH
N6
1 N
N
R
R2
=
7
OH
R2
Designed series
or other
secondary
amines
Overlay of adenosine
and designed scaffold
OH
Figure 2. Design of 7-substituted thiazolo[5,4-d]pyrimidines as potential
adenosine A2A agonists. The 2-symmetry axis considered for the molecular
superposition of adenosine and thiazolo[5,4-d]pyrimidine scaffolds in the
A2AAR binding site is shown.
According to this superposition model, the R2 substituent
overlays with the ribose of adenosine, and the R, matches the C-2
substituents in adenosine derivatives. In our case, R is a 2-
arylamino mimicking the C2 of agonist 4, and according to the
docking model it occupies the same extracellular region than the
C2-substituent of the crystalized agonist 525 and the phenylethyl
group of antagonist ZM241385 (Figure 3).26 Pooling together the
two parallel docking runs (i.e. considering the active and inactive-
like conformation of the receptor), this conserved binding mode
was found among the top three ranking poses in more than 78% of
the compound series. In this pose, the ligand is anchored by a
hydrogen bond interaction between the conserved Asn2536.55 and
N4 of the ligand scaffold, allowing the R and R2 substituents to be
positioned as described above. It has been well proven that, in the
active-like structures, the binding site of the ribose corresponds to
an otherwise highly hydrated region in the antagonist-bound
structures. Interestingly, R2 in many of our compounds displaces
some of the water molecules present within this area in the
corresponding X-ray structures. In particular, the compounds
1
compounds were characterized by using H NMR, 13C NMR and
HR-ESIMS analysis. Four series of compounds (as depicted in
Scheme 1) were prepared in order to understand the structure-
activity relationships.
Cl
Cl
N
H
14
+
N
NH2
Cl
R1
N C S
N
+
N
S
N
S
N
N
NH
12
NH
b
a
N
N
R1
N
R1
11
13
8-10
R2
X
Cl
N
R2
N
R2
N
N
R2
N
N
N
N
N
S
N
N
NH
N
S
N
N
NH
NH
NH
S
R1
R1
S
R1
N
R1
13a-i
(series 1)
8
9
10
(series 4)
(series 2)
(series 3)
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Scheme 1. Synthesis of 7-substituted thiazolo[5,4-d]pyrimidines 8-10,
13.Reagents and conditions: (a) KF/alumina (20 mol%), ACN, 60 °C, 5 h;
(b) MeOH, rt, 4-5 h.
receptors (3-7 fold) than compound 10m (> 100 and 12 fold,
respectively).
1
2
3
4
5
6
7
8
9
Characterization of selected compounds in A2AAR
functional assay. Based on the predictions of molecular
modeling, we investigated the potential presence of agonistic
activity of the prolinol linked analogs. Six compounds bearing a
hydroxymethylene substituent on the pyrrolidine (compounds
10b, 10d, 10g, 10j, 10l, and 10n) were tested in an A2AAR
functional assay consisting of the measurement of adenylyl
cyclase activity. Their corresponding analogs without the
hydroxymethylene substituent (compounds 10a, 10c, 10f, 10i, 10k
and 10m) were also tested, the hypothesis being that they would
not show agonistic activity as opposed to their hydroxymethylene
substituted analogs. The results of the functional assay are shown
in Figure 4.
Interestingly, when benzyl isothiocyanate was reacted with 6-
dichloro-5-aminopyrimidine (11), two products 13j and 13k were
formed in 5% and 52% yield, respectively. The disubstituted
product 13k was formed via thioacylation of NH of the product
13j.20 The formation of products 13j and 13k is depicted in
Scheme 2. The exact structures of all synthesized compounds are
shown in Tables 1-4.
Cl
Cl
Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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37
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42
43
44
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S
N
NH2
Cl
N
S
N
N
a
NCS
NH
N
S
NH
N
N
N
N
11
12
13j
13k
Scheme 2. Reaction of 6-dichloro-5-aminopyrimidine (11) with benzyl
isothiocyanate (12): (a) KF/alumina (20 mol%), ACN, 60 °C, 5 h.
Adenosine receptor binding studies. Radioligand binding assays
were performed for all four series of thiazolo[5,4-d]pyrimidines at
the human A1-, A2A-, and A3AR subtypes. Due to the lack of a
suitable radioligand for A2B adenosine receptors, the effect on
NECA-stimulated adenylyl cyclase was determined as a measure
for A2B affinity. None of the compounds in this study showed
detectable affinity (Ki values > 10,000 nM) for the A2B subtype
(data not shown). Most compounds of the series 1 bearing a 7-
chloro substituent showed modest affinity for A2AAR (Table 1).
For the best compound 13h, an A2A Ki of 1,050 nM was
determined. For thiazolo[5,4-d]pyrimidines containing an
alkylamino substitution at C-7 position (series 2), similar results
were found (Table 2) with compound 8b showing the highest
binding affinity for A2AAR (Ki = 1,270 nM). In the third series,
comprising a six-membered saturated N-heterocycle at C-7
position, a morpholine substituted analog 9a showed good binding
affinity towards adenosine A2A receptor with Ki value of 333 nM,
and >3,000 and 13-fold selectivity towards A1 and A3 receptors,
respectively (Table 3). Compounds from the fourth series where
the C-7 position of the thiazolo[5,4-d]pyrimidine scaffold is
R
N
R
N
R
N
N
N
N
N
N
N
NH
NH
NH
S
S
S
N
N
N
Cl
10f: R = -H
10a: R = -H
10c: R = -H
10g: R = -CH2OH
10b: R = -CH2OH
10d: R = -CH2OH
R
N
R
R
N
N
N
N
N
N
N
NH
N
NH
NH
F
substituted with
a five-membered saturated N-heterocycle,
S
N
S
N
S
N
OMe
OMe
showed promising binding affinity for the adenosine A2A receptor
(Table 4). The most potent compound in this series is 10m
Br
F
MeO
bearing
a 3,4,5-tri-OMe phenylamino group at C-2 and
10k: R = -H
10l: R = -CH2OH
10m: R = -H
10n: R = -CH2OH
10i: R = -H
10j: R = -CH2OH
pyrrolidine at C-7 position. Compound 10m showed binding
affinity for A2AAR with Ki value of 102 nM. The binding affinity
(Ki) of this compound for A1 and A3ARs was >100,000 and 1,280
nM, respectively, indicating its selectivity towards A2AAR.
Figure 4. Agonistic activity of prolinol analogs 10b, 10d, 10g, 10j, 10l,
10n and their corresponding pyrrolidine (non-prolinol) analogs 10a, 10c,
10f, 10i, 10k, 10m at A2A adenosine receptors.
All predicted agonists 10b, 10d, 10g, 10j, 10l, and 10n showed
about 50-60% efficacy at 100 M concentration (reference value
The obtained results for A2AAR binding allowed to envisage a
clear structure-activity relationship. The analogs substituted with
electron-donating groups (EDG) at the C-2 phenylamino ring
showed higher binding affinity towards A2AAR than analogs with
electron-withdrawing groups (EWG) at the phenyl ring. In
agreement with this, compound 13h in series 1 substituted with a
3,4,5-trimethoxy phenyl ring shows the highest affinity with a Ki
value of 1,050 nM for the A2A adenosine receptor, whereas analog
13e substituted with p-nitrophenyl ring is inactive (Ki > 30,000)
(Table 1). Similar observations were also made in series 4, where
10m substituted with an EDG shows better binding affinity (Ki =
102 nM) than 10h possessing an EWG (Ki = 3,180 nM).
Furthermore, among various substituents (viz. chloro,
dialkylamino, six-membered N-heterocycle, and five-membered
N-heterocycle) attempted at C-7 position, particularly the analogs
bearing a five-membered N-heterocycle at C-7 position presented
with the highest affinity. The best compound from this series is
10m (Ki for A2AAR is 102 nM). The five-membered N-
heterocycle bearing compounds 10b and 10l also exhibited high
A2Aaffinity; however, they were less selective against A1 and A3
for NECA
= 100%), whereas the corresponding control
compounds 10a, 10c, 10f, 10i, 10k and 10m showed neglegible or
significantly lower (10-40%) efficacy levels. Compound 10m,
with the highest A2A affinity of all new ligands reported in this
study, presented the lowest A2A efficacy (10%, see Figure 4).
However, its corresponding prolinol analog 10n, with a 3-times
lower affinity, showed >50% efficacy, substantiating the
relevance of a hydroxymethylene substituent on the pyrrolidine
for agonistic activity. In all the six pair of analogs (10a/10b,
10c/10d, 10f/10g, 10i/10j, 10k/10l, 10m/10n) tested, the 2-
hydroxymethylene substituted compounds consistently show
higher efficacy than their analogs lacking this functional group.
Taken together, this clearly indicated that the presence of 2-
hydroxymethylene group on pyrrolidine (i.e. prolinol) is
relevantly contributing to A2AAR agonism. The prolinol
containing compounds 10b and 10l were the most potent A2A
adenosine receptor partial agonists in this study with Ki values of
200 and 153 nM.
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R2
X
Cl
N
Cl
N
R2
N
R2
Cl
N
1
2
3
4
Cl
N
N
N
R2
N
N
S
N
N
N
N
S
N
N
Ph
N
N
S
NH
N
S
N
N
S
NH
O
N
N
S
N
S
NH
N
R1
NH
N
Ph
NH
NH
NH
S
N
Ph
13a-h
13i
13k
13l
8a-d
9a-i
10a-n
O
R1
5
R1
R1
6
7
8
Table 1. Adenosine receptor binding affinity of 7-chloro substituted thiazolo[5,4-d]pyrimidines 13a-l (series 1)
Ki in nMa
Entry
R1
A1
A2A
A3
9
13a
13b
13c
13d
13e
13f
13g
13h
13i
H
4-Cl
4-OMe
4-Me
4-NO2
2,6-di-F
3-Br
3,4,5-tri-OMe
> 100,000
1,520 (1,130-1,760)
2,690 (1,750-4,130)
3,570 (2,140-5,960)
4,930 (2,580-9,410)
>30,000
8,110 (7,690-8,560)
2,960 (2,600-3,370)
1,050 (1,030-1,080)
1,500 (1,420-1,590)
1,870 (1,290-2,720)
>100,000
3,670 (3,030-4,440)
4,940 (3,240-7,510)
5,880 (4,980-6,950)
4,640 (3,540-6,080)
7,950 (4,900-12,900)
17,200 (12,500-23,700)
3,900 (2,970-5,120)
2,650 (2,260-3,110)
7,330 (5,920-9,090)
1,030 (905-1,170)
>100,000
25,000 (13,900-44,700)
11,500 (10,200-12,900)
>100,000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>30,000
17,800 (15,400-20,600)
12,600 (9,550-16,600)
>100,000
7,240 (5,610-9,330)
>30,000
-
-
-
13k
13l
>100,000
Table 2. Adenosine receptor binding affinity of 7-dialkylamino-substituted thiazolo[5,4-d]pyrimidines 8a-d (series 2)
Ki in nMa
Entry
R1
R2
A1
A2A
A3
8a
8b
8c
8d
H
H
4-Cl
4-Cl
Et
Me
Et
> 100,000
> 100,000
16,500 (9,900-27,500)
> 100,000
2,210 (1,100-4,440)
1,270 (927-1,730)
11,200 (8,590-14,500)
>100,000
> 30,000
1,280 (1,260-1,310)
3,890 (3,160-4,790)
> 10,000
Me
Table 3. Adenosine receptor binding affinity of thiazolo[5,4-d]pyrimidines 9a-i substituted with six-membered aliphatic N-heterocycle at C-7 position
(series 3)
Ki in nMa
A2A
333 (276-402)
980 (593-1,620)
2,840 (2,490-3,240)
9,090 (6,860-12,000)
8,390 (8,030-8,750)
Entry
R1
X-R2
A1
A3
9a
9b
9c
9d
9e
-H
-H
-H
-H
-H
O
> 100,000
> 30,000
> 30,000
> 30,000
> 100,000
4,460 (2,950-6,760)
12,100 (10,100-14,400)
4,310 (2,600-7,150)
> 10,000
CH2
N-Me
NH
-NEt
> 30,000
N
9f
-H
> 100,000
>100,000
> 100,000
9g
9h
9i
4-Cl
4-Cl
4-Cl
O
> 100,000
> 100,000
> 100,000
1,400 (1,070-1,830)
13,200 (9,840-17,800)
>100,000
> 100,000
18,300 (16,500-20,300)
> 10,000
CH2
N-Me
Table 4. Adenosine receptor binding affinity of thiazolo[5,4-d]pyrimidines 10a-n substituted with five-membered aliphatic N-heterocycle at C-7
position (series 4)
Ki in nMa
Entry
R1
R2
A1
> 30,000
555 (448-686)
> 100,000
629 (517-764)
713 (637-799)
>100,000
A2A
A3
10a
10b
10c
10d
10e
10f
-H
-H
H
351 (266-462)
200 (190-210)
2,060 (1,750-2,430)
466 (422-515)
1,110 (743-1,660)
974 (714-1,330)
313 (273-360)
8,630 (6,380-11,700)
978 (825-1,160)
4,260 (4,020-4,500)
934 (740-1,180)
594 (431-819)
563 (360-879)
843 (685-1,040)
-CH2OH
H
-CH2OH
-CH2OH
-H
4-Cl
4-Cl
4-OMe
4-Me
4-Me
10g
-CH2OH
399 (363-439)
10h
4-NO2
-H
>30,000
3,180 (1,850-5,440)
3,630 (2,590-5,080)
10i
2,6-Di-F
2,6-Di-F
3-Br
-H
2,310 (2,070-2,570)
7,970 (6,890-9,220)
>100,000
530 (328-856)
>100,000
2,260 (2,050-2,500)
3,260 (2,680-3,970)
245 (200-300)
153 (117-200)
102 (84-125)
3,190 (2,710-3,740)
3,420 (2,560-4,580)
1,290 (782-2,130)
1,070 (919-1,260)
1,280 (1,010-1,630)
1,460 (900-2,380)
10j
10k
10l
10m
10n
-CH2OH
-H
-CH2OH
-H
3-Br
3,4,5-tri-OMe
3,4,5-tri-OMe
-CH2OH
974 (534-1,780)
330 (288-378)
aShown are Ki values in nM with 95% confidence limits in parentheses
These experimental results are in line with the molecular
mechanism proposed for these compounds as being (partial)
agonists (see Figure 3), i.e. interactions of the hydroxyl group in
prolinol-containing compounds with His7.43 (or alternatively
Ser7.42) can, to some extent, stabilise the active conformation of
the receptor.
Aqueous solubility and lipophilicity determination of
promising compounds. The thermodynamic equilibrium
solubility of selected compounds viz. 10b, 10f, 10g, 10k, 10l and
10m was investigated in water, PBS, SGF and SIF (results are
shown in Table S1). Amongst these compounds, the 7-prolinol-2-
phenylamino linked thiazolo[5,4-d]pyrimidine 10b displayed
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Journal of Medicinal Chemistry
excellent solubility (4.3, 4.8, and 3.5 mg/ml, respectively) in
General method for preparation of compounds 8a-d, 9a-i
and 10a-n.30 To the solution of 2-amino-7-thiazolo[5,4-
d]pyrimidines (13a-k, 0.25 mmol) in MeOH (3 mL) was added
secondary amines (1.0 mmol). The mixture was stirred at room
temperature for 12-24 h and then concentrated under reduced
pressure. The reaction mixture was then dried under vacuum and
the final products 8a-d, 9a-i and 10a-n were recrystallized with
methanol and chloroform mixture giving 85-98% isolated yield.
The spectral data of a representative compound 10b is provided
here. Spectral data of all remaining compounds is provided in
supporting information.
1
2
3
4
5
6
7
8
water, PBS and SGF. Other compounds 10f, 10k and 10l also
showed good water solubility with solubility values >150 µg/ml.
All compounds were poorly soluble in SIF. The Log P and Log D
values of these compounds were also determined using the n-
octanol-water partition coefficient method and results are listed in
Table S1. In line with solubility values, compound 10b showed
better Log P and Log D values than other compounds
Conclusion
In summary, through
a rational computer-aided design
9
approach, we have discovered 7-prolinol-substituted thiazolo[5,4-
d]pyrimidines as potent and selective A2A receptor partial
agonists. Importantly, this novel chemotype is one of the few
examples of A2A adenosine receptor agonists not based on the
adenosine scaffold. Compounds 10b and 10l emerge as the most
potent adenosine A2AAR partial agonists reported here, with Ki
values of 200 and 153 nM, also showing moderate selectivity for
A2A over A1 and A3 receptors. In addition, compound 10b showed
excellent solubility in water, PBS and SGF. This study has
demonstrated that molecular modeling predictions can be used to
successfully design novel A2A adenosine receptor agonists whose
basic structure does not rely on the adenosine scaffold. This
unique new class of A2A partial agonists can be an excellent
starting point in the design of novel agents for the treatment of
important diseases such as cerebral and cardiac ischemia,
epilepsy, thrombosis, and arterial hypertension.
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2-(Phenylamino)-7-[2(S)-hydroxymethyl-pyrrolidin-1-yl]-
thiazolo[5,4-d]pyrimidine (10b). Yield: 86%; brown solid; m.p.
1
232-234 ºC; H NMR (500 MHz, DMSO-d6, ppm):  10.51 (s,
1H, NH), 8.17 (s, 1H), 7.72 (d, 2H, J = 7.6 Hz), 7.36 (d, 2H, J =
7.5 Hz), 7.00 (t, 1H, J =7.3 Hz), 4.82 (t, 2H, J = 4 Hz), 3.72-3.70
(m, 2H), 2.09-1.93 (m, 5H); 13C NMR (125 MHz, CD3OD, ppm):
δ 159.84 (C), 158.01 (C), 153.25 (C), 152.14 (CH), 141.77 (C),
130.14 (CH), 124.53 (C), 123.70 (CH), 119.24 (CH), 64.42 (CH2),
64.42 (CH2), 61.51 (CH), 30.82 (CH2), 28.82 (CH2); HR-ESIMS:
m/z calcd 328.1228 for C16H17N5OS+H+ (328.1227).
Radioligand binding to hA1, A2A and A3ARs. [3H]CCPA (1
nM), [3H]NECA (10 nM), and [3H]HEMADO (1 nM) were
utilized in radioligand binding assays to membranes prepared
from CHO cells expressing recombinant hA1, hA2A and hA3 ARs.
All procedures were carried out as previously described in
detail.13, 35 Non-radioactive compounds were initially dissolved in
DMSO and diluted with buffer to the final concentration where
the amount of DMSO never exceeded 1%. For incubation 96-well
microplates with filter bottoms (Millipore MultiScreen MAFC)
were used and incubation was terminated after 3 h at 25 °C by
filtration through the built-in filter bottoms. The wells were rinsed
three times with 200 µL of ice-cold binding buffer. Filter plates
were dried and counted in a WallacMicroBeta Counter after
addition of 20 µL of scintillator. Ki values were calculated with
the program Prism (GraphPad Software Inc., La Jolla, CA).
Experimental section
Molecular modeling studies.The structures of A2AAR in both
active-like (PDB: 2YDO, in complex with adenosine)28 and
inactive conformations (PDB: 4EIY, in complex with antagonist
ZM241385),26 were prepared for docking experiments. Selected
crystallographic waters occupying the binding site were retained
and the fragments for crystallization purposes removed. The
structures were then refined in order to model the missing loops
and add protons, as described earlier.31 Special attention was paid
to the protonation state of histidine residues in the binding site,
His6.52250 and His7.43278, which were both modeled as neutral
and protonated in H, to allow the characteristic interactions of
agonists according to the A2A-agonist crystal structures.25, 27, 28 All
compounds were built and energy minimized using Maestro
software32 and the OPLSAA force-field.33 Each ligand was
docked 20 times with default (high accuracy) genetic algorithm
(GA) search parameters, using the scoring function Chemscore as
implemented in GOLD34 and allowing full flexibility for the
ligand. The search sphere was centered on the side chain (CG1) of
Ile7.39 and expanded with a radius of 15 Å, thus ensuring a
generous enough search space comprising the binding site
experimentally. The criterion for the docking posess election was
based on a combination of the Chemscore ranking and the
population (convergence) of the solutions according to anr.m.s.d.
clustering criteria of 1 Å.
General method for preparation of 2-amino-7-
chlorothiazolo[5,4-d]pyrimidines 13a-k.30 To the solution of
4,6-dichloro-5-aminopyrimidine (11, 164 mg, 1 mmol) in ACN (4
mL) was added KF/alumina (20 mol%) followed by
isothiocyanate 12 (1 mmol). The mixture was stirred at 60 °C for
5 h, then cooled to room temperature and concentrated under
reduced pressure. The resulted mixture was dissolved in 10%
methanol in CHCl3 and the catalyst KF/alumina was filtered and
washed with acetone and then dried and reused. The filtrate was
dried and washed with hexane: diethyl ether mixture to get
products 13a-k in 80-96% yield.
Measurement of adenylyl cyclase activity. The agonistic
activity of compounds at the A2AAR was determined in adenylyl
cyclase experiments as described previously.13 To study the
interaction of compounds with A2B adenosine receptors inhibition
of NECA-stimulated adenylyl cyclase activity was measured.
AUTHOR INFORMATION
Corresponding authors
*Tel. +91-191-2585006 (extn. 345); Fax: +91-191-2585333; E-
mail: sbharate@iiim.ac.in (S.B.B.)
*Tel. 46(0)18-471-5056; E-mail: hugo.gutierrez@icm.uu.se
(H.G.T.)
Author Contributions. S.B.B. designed all chemistry strategies,
synthetic protocols, and coordinated whole study; B.S.
synthesized all compounds; S.K. performed adenosine receptor
binding assays; A.O. performed molecular modelling studies;
V.K. and S.S.B. performed solubility and Log P determinations;
S.S.B. analyzed and interpreted solubility/Log P results; K.N.K.
evaluated and interpreted adenosine receptor binding data; HGT
interpreted molecular modelling results; S.B.B., R.A.V., H.G.T.,
K.N.K. contributed to manuscript writing.
ACKNOWLEDGEMENT
B.S. and S.S.B. thanks ICMR for SRF and RA fellowships,
respectively. VK thanks UGC for fellowship. Authors are
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17. Diedrichs, N.; Henninger, K.; Hubsch, W.; Kramer, T.; Krahn, T.;
Rosentreter, U.; Shimada, M.; Stasch, J. P. Substituted 2-thio-3,5-
dicyano-4-phenyl-6-aminopyridines and their use as adenosine
receptor-selective ligands. WO2003008384 A1 2003.
thankful to analytical department, IIIM for analytical support. The
financial support from CSIR 12th FYP grant BSC-0205 is
gratefully acknowledged.
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18. Beukers, M. W.; Chang, L. C.; von Frijtag Drabbe Künzel, J. K.;
Mulder-Krieger, T.; Spanjersberg, R. F.; Brussee, J.; IJzerman, A. P.
New, non-adenosine, high-potency agonists for the human adenosine
A2B receptor with an improved selectivity profile compared to the
reference agonist N-ethylcarboxamidoadenosine. J. Med. Chem.
2004, 47, 3707-3709.
19. Rodríguez, D.; Gao, Z.-G.; Moss, S. M.; Jacobson, K. A.; Carlsson,
J. Molecular docking screening using agonist-bound GPCR
structures: Probing the A2A adenosine receptor. J. Chem. Inf. Model.
2015, 55, 550-563.
ABBREVIATIONS
A2AAR, Adenosine A2A receptor; cAMP, cyclic adenosine
monophosphate; CCPA, 2-chloro-N6-cyclopentyladenosine; EDG,
electron-donating groups;EWG, electron-withdrawing groups;
FDA, Food and Drug Administration; GPCR, G protein-coupled
receptor;
HEMADO,
2-hexyn-1-yl-N6-methyladenosine;
HENECA, 2-hexynyl-N-ethylcarboxamidoadenosine; NECA, N-
ethylcarboxamidoadenosine; PBS, phosphate buffer saline; SGF,
simulated gastric fluid; SIF, simulated intestinal fluid.
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20. Liu, J.; Patch, R. J.; Schubert, C.; Player, M. R. Single-step
syntheses
of
2-amino-7-chlorothiazolo[5,4-d]pyrimidines:
SUPPORTING INFORMATION AVAILABLE: Experimental
details and NMR spectra scans. This material is available free of
charge via the Internet at http://pubs.acs.org.
intermediates for bivalent thiazolopyrimidines. J. Org. Chem. 2005,
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21. Legraverend, M. Recent advances in the synthesis of purine
derivatives and their precursors. Tetrahedron 2008, 64, 8585–8603.
22. Barodia, S. K.; Mishra, C. B.; Prakash, A.; Senthil Kumar, J. B.;
Kumari, N.; Luthra, P. M. Novel 8-(furan-2-yl)-3-benzyl thiazolo
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10b (green) with A2A
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