Studies on enantioselectivity of chiral 4-acetylamino-6-alkyloxy-2-alkylthiopyrimidines acting as antagonists of the human A3 adenosine receptor

Article abstract of DOI:10.1039/c7md00375g

Three A₃ adenosine receptor (AR) antagonists (1-3) selected from 4-acylamino-6-alkyloxy-2-alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or thioether side chains. These compounds gave

Full text of DOI:10.1039/c7md00375g

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RESEARCH ARTICLE
Studies on enantioselectivity of chiral
4-acetylamino-6-alkyloxy-2-alkylthiopyrimidines
acting as antagonists of the human A3 adenosine
receptor†
Cite this: DOI: 10.1039/c7md00375g
a
Barbara Cosimelli, a Giovanni Greco,
a Sonia Laneri, a Ettore Novellino,
*
c
Antonia Sacchi, a Simona Collina, b Daniela Rossi, b Sandro Cosconati,
d
Elisabetta Barresi, d Sabrina Taliani, d Maria Letizia Trincavelli
d
and Claudia Martini
Three A3 adenosine receptor (AR) antagonists (1–3) selected from 4-acylamino-6-alkyloxy-2-
alkylthiopyrimidines previously investigated by us were modified by inserting a methyl group on their ether or
thioether side chains. These compounds gave us the chance to evaluate whether their higher lipophilicity, re-
duced conformational freedom and chirality might improve the potency towards the A3 AR. Racemic mix-
tures of 1–3 were resolved using chiral HPLC methods and the absolute configurations of the enantiomers
were assigned by chiroptical spectroscopy and density functional theory calculations. We measured the affin-
ity for human A1, A2A, A2B and A3 ARs of the racemic mixtures and the pure enantiomers of 1–3 by radioligand
competition binding experiments. Cell-based assays of the most potent enantiomers confirmed their A3 AR
antagonist profiles. Our research led to the identification of (S)-1 with high potency (0.5 nM) and selectivity
as an A3 AR antagonist. Moreover we built a docking-model useful to design new pyrimidine derivatives.
Received 24th July 2017,
Accepted 8th November 2017
DOI: 10.1039/c7md00375g
rsc.li/medchemcomm
growth.3 Borea P. A. et al.4 have recently outlined the current
and potential clinical applications of selective agonists and
Introduction
Adenosine is ubiquitously located in mammalian organisms,
where it acts as an agonist of four subtypes of G-protein-
coupled-receptors (GPCRs), namely the A1, A2A, A2B and the A3
adenosine receptors (ARs).1,2 Activation of ARs leads to differ-
ent intracellular events related to inhibition (A1 and A3) or
stimulation (A2A and A2B) of adenylate cyclase, stimulation of
phospholipase C (A1, A2B, and A3), activation of potassium
channels and inhibition of calcium channels (A1).2 ARs are
considered to be promising pharmacological targets given
their key role in important physiological and pathological pro-
cesses. The A3 AR is involved in cell cycle regulation and cell
antagonists of this receptor. As detailed in the above quoted
review, agonists can be employed as cardioprotective,
cerebroprotective and anti-inflammatory agents, whereas an-
tagonists might be useful for the treatment of stroke, glau-
coma, asthma and allergies.
The pyrimidine ring represents one of the heterocyclic
scaffolds featured by potent antagonists of the human A3
AR,5–8 including some 4-acylamino-6-alkyloxy-2-alkylthio-
pyrimidines of general formula I (Chart 1, where R, R′ and R″
are alkyl groups) reported by us some years ago.7
Recently, we have focused our attention on compounds Ia
and Ib of series I (Chart 1) as suitable structures to continue
our research in this field. Particularly, we decided to prepare
and test 1 as an α-methyl derivative of Ia together with 2 and
3 as α-methyl derivatives of Ib to assess whether the designed
compounds, being more lipophilic and less flexible than
their desmethyl counterparts, might exhibit improved po-
tency as A3 AR antagonists. Moreover, as α-methylation of Ia
and Ib introduces a stereogenic center on the ether (1 and 3)
or the thioether (2) side chain, we had a chance to study the
potential influence of chirality on the interaction of these py-
rimidine derivatives with the target receptor. To our knowl-
edge, only a few investigations on chiral A3 AR antagonists
a Dipartimento di Farmacia, Università di Napoli “Federico II”, Via Domenico
Montesano 49, 80131 Naples, Italy. E-mail: giovanni.greco@unina.it;
Fax: +39 081 678107; Tel: +39 081678645
b Dipartimento del Farmaco, Università di Pavia, Viale Torquato Taramelli 12,
27100 Pavia, Italy
c Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche,
Università degli Studi della Campania “Luigi Vanvitelli”, Via Antonio Vivaldi 43,
81100 Caserta, Italy
d Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126
Pisa, Italy
† Electronic supplementary information (ESI) available: Chemistry and biology
experimental sections for the described compounds. See DOI: 10.1039/
c7md00375g
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Chart 1 Design of chiral pyrimidines 1–3 by formal methylation of Ia and Ib.
have been so far reported in the literature. Specifically, chiral
1,4-dihydropyridines have been investigated by van Rhee, A.
M. et al.9 as well as by Jiang J.-L. et al.,10 whereas Jacobson, K.
A. et al. described carbocyclic adenosine analogues character-
ized as A3 AR adenosine antagonists and partial agonists.11
In our work the racemic mixtures of the novel pyrimidine
derivatives were resolved using chiral HPLC methods and the
absolute configurations of the resulting pure enantiomers
were assigned by a combined approach of chiroptical spectro-
scopy and density functional theory (DFT) calculations. The
results of these studies have been recently published.12
In the present work, racemic mixtures and the pure
enantiomers of 1–3 were assayed in competition binding ex-
periments to measure their affinities for the human A3 AR
and their selectivity trend towards the other AR subtypes.
Cell-based functional experiments were carried out on the
most potent enantiomers to confirm the expected antagonist
efficacy profile. Finally, in silico docking studies using a 3D
model of the human A3 AR were performed to hypothesize
the interactions of the most potent enantiomers with the re-
ceptor binding site at the molecular level. This paper de-
scribes the results of our studies.
alkylated to give 8–10. When the reactant was 6 the reaction
gave a mixture of isomers 9 and 9a. As a last step, the
4-amino group of 8–10 was converted into the amide group
of 1–3 by reaction with acetic anhydride and concentrated
H2SO4 as a catalyst.
As recently reported,12 the resolution of racemic 1–3 by
semi-preparative HPLC using chiral stationary phases was
performed to isolate each enantiomer in small amounts suit-
able for configurational assignment and biological
investigations.
Results
Chemistry
Derivatives 1–3 were obtained in three steps starting from the
commercially
available
4-amino-6-hydroxy-2-mercapto-
pyrimidine monohydrate (4) as shown in Scheme 1 (addi-
tional experimental details for each step including characteri-
zation of the intermediates can be found in the ESI† of this
publication). Alkylation of the sulfur atom at the 2-position
by reaction with the opportune bromoalkane in 1 M NaOH
gave derivatives 5–7 which were collected by filtration and
resulted in sufficiently pure compounds to be subsequently
used without further purification (5 and 7 were previously
published).7 As a second step, the 4-oxygen atom of 5–7 was
Scheme 1 Reagents: (a) bromopropane, (1-bromoethyl)benzene or
benzylbromide, NaOH 1 M, 50–55 °C, 15–90%. (b) (1-Bromoethyl)-
benzene, bromopropane or 2-bromobutane, K2CO3, CH3CN, reflux,
60–75%. (c) (CH3CO)2O, H2SO4 conc. (cat.), 50–60 °C, 41% (1) or 52%
(2) or 70% (3).
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Briefly, a standard screening protocol was firstly applied
by using an analytical Chiralcel OJ-H column (4.6 mm × 150
ARs (Ki values greater than 1000 nM; their percentages of in-
hibition of specific radioligand binding determined at 10 μM
are listed in Table 1S of the ESI†). On the basis of these re-
sults, enantiomeric 1–3 were tested for their binding affini-
ties exclusively for the A3 AR following the same procedures
employed for the racemic mixtures.
mm,
5 μm), whose chiral selector is cellulose tris-(4-
methylbenzoate) coated on a silica gel substrate. Baseline sep-
aration of 2 and 3 was achieved by eluting with pure
methanol.
As regards 1, high enantioselectivity and resolution were
achieved by using heptane/ethanol 95/5 (v/v) as the eluent.
These experimental conditions were adopted for the chro-
matographic scale-up so as to afford enantiomeric 1–3 in
good yields (82%, 93% and 97%, respectively) and with an
optical purity above 99% suitable for both configurational
studies and biological investigations.
Absolute configurations of enantiomeric 1–3 were
assigned by comparing experimental and calculated optical
rotatory dispersion curves and, in the case of enantiomeric 1
and 2, also comparing the electronic circular dichroism and
vibrational circular dichroism spectra, with DFT calculations.
According to our studies, the S absolute configuration was
assigned to the levo isomers of 1–3 and the R absolute config-
uration to the corresponding dextro isomers (Chart 2).
The antagonist efficacies of the enantiomers exhibiting
the highest affinities for the A3 AR, namely eutomers (S)-1,
(S)-2 and (S)-3, were evaluated by assessing their abilities to
counteract
2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyl-
carboxamide (Cl-IB-MECA)-mediated inhibition of cAMP accu-
mulation produced by forskolin (FK). Cl-IB-MECA is a selec-
tive A3 AR agonist whereas FK is a non-selective activator of
adenylate cyclase. The obtained data are expressed as IC50
values (concentrations inhibiting the effect of the selective
Cl-IB-MECA by 50%).14 Intracellular cAMP levels were mea-
sured as previously described.15 (Methods are detailed in the
ESI†).
Table 1 lists the binding affinities of 1–3, tested as race-
mic mixtures or enantiomers, for the human A3 AR expressed
as Ki values. The binding affinities for the same receptor of
the previously described pyrimidine derivatives Ia and Ib7 are
also given for comparison purposes. As mentioned, the affini-
ties of the new compounds for the A3 AR range from
subnanomolar to micromolar values. The concentration–re-
sponse curves of the tested compounds are depicted in
Fig. 1, whereas their antagonist potencies, expressed as IC50
values, are listed in Table 1.
Biology
The affinities of 1–3 for human ARs, tested as racemic mix-
tures, were evaluated by radioligand competition binding ex-
periments which assessed their ability to displace [3H]8-
cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) from the human
A1 AR or [3H]5′-N-ethylcarboxamidoadenosine ([3H]NECA)
from the human A2A, A2B and A3 ARs expressed in Chinese
hamster ovary (CHO) cells.13 In all binding assays, com-
pounds 1–3, similarly to their desmethyl counterparts Ia and
Ib, exhibited high or moderate potency for the A3 AR,
whereas they lacked appreciable affinity for A1, A2A and A2B
All the three selected compounds acted as antagonists of
the A3 AR with potencies comparable to the affinities
obtained in radioligand binding experiments.
Discussion
Insertion of an α-methyl group on the benzyloxy side chain
of Ia to yield 1 increases potency. In fact, (S)-1 and (R)-1 are
more potent than their desmethyl counterparts, by 15-fold
and 4.4-fold, respectively. These findings suggest that the
α-methyl group helps the PhCHIJCH3)O substituent of both
(S)-1 and (R)-1 to optimize their interactions within a specific
region of the human A3 AR binding site. This result might be
achieved by each of the two ligands through conformational
effects (i.e. reduced torsional freedom) and/or a direct hydro-
phobic interaction with the lipophilic side chain residues of
the receptor.
Conversely, α-methylation of the benzylthio side chain of
Ib to give 2 does not significantly affect the potency, the race-
mic mixture and the corresponding enantiomers being nearly
equipotent with the desmethyl parent structure.
Such data indicate that this type of α-methylation some-
how leaves the interactions between Ib and the A3 AR almost
intact.
When a methyl group is inserted onto the α-carbon of the
n-propyloxy side chain of Ib to yield 3, we observe contrasting
effects on the potency depending on the chirality. Specifi-
cally, compared with the desmethyl parent compound, (S)-3
Chart 2 Structures of the enantiomers of 1–3 represented with their
absolute configurations.
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Table 1 Binding affinities, expressed as Ki values, of 4-acetylamino-6-alkyloxy-2-alkylthiopirimidine 1–3 for the human A3 AR tested as racemic mixtures
or pure enantiomers. Antagonist potencies of (S)-1, (S)-2 and (S)-3 for the human A3 AR are expressed as IC50 values
No
R
R′
Ki [nM]a
IC50 [nM]a
b
c
hA3
hA3
Iad
CH3CH2CH2
C6H5CH2
C6H5CH2
CH3CH2CH2
7.5 0.22
525 21
Ibd
(R/S)-1
(R)-(+)-1
(S)-(−)-1
(R/S)-2
(R)-(+)-2
(S)-(−)-2
(R/S)-3
(R)-(+)-3
(S)-(−)-3
DPCPX
NECA
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
C6H5IJCH3)CH
C6H5IJCH3)CH
C6H5IJCH3)CH
C6H5CH2
C6H5IJCH3)CH
C6H5IJCH3)CH
C6H5IJCH3)CH
CH3CH2CH2
CH3CH2CH2
CH3CH2CH2
CH3CH2IJCH3)CH
CH3CH2IJCH3)CH
CH3CH2IJCH3)CH
0.6 0.04
1.7 0.4
0.5 0.2
580.1 70.3
737.5 28.5
440.2 55.2
195.1 32.4
1390 187
183.2 44.1
>1000
7.9 0.9
428.2 36.9
152.9 14.3
C6H5CH2
C6H5CH2
75.5 5.2
0.2 0.1
Cl-IB-MECA
a
The Ki and IC50 values are means SEM of at least three determinations derived from an iterative curve-fitting procedure (Prism program,
GraphPad, San Diego, CA). Displacement of specific [3H]NECA binding in membranes obtained from CHO cells stably expressing the human
A3 AR. The IC50 values correspond to the concentrations inhibiting the effect of the selective A3 AR agonist Cl-IB-MECA by 50%. Data from
ref. 7, assayed with a purity of 96%.
b
c
d
is more potent by 2.9-fold, whereas (R)-3 is less potent by 2.6-
fold. In this case, α-methylation modifies the conformational
properties of the s-butyloxy side chain of the eutomer and of
the distomer so as to enhance or, respectively, reduce ligand–
receptor attractive interactions.
scaffold of (S)-1 and (R)-1 engages in π-stacking and van der
Waals interactions with the F168 phenyl ring on one side and
Docking studies (see the ESI† for methods) carried out on
the most potent enantiomers, namely, (S)-1 and (R)-1, suggest
that these compounds form a double-bridged H-bond between
the ligand N3–C4–NH fragment and the receptor N250 side
chain (Fig. 2). Such an interaction is considered to be funda-
mental for tight ligand binding to both A3 and A2A ARs.16,17 By
looking at Fig. 2, it can also be noticed that the pyrimidine
Fig.
1 Effects of (S)-1, (S)-2 and (S)-3 on Cl-IB-MECA-mediated
inhibition of cAMP accumulation in human A3 AR-transfected CHO
cells. CHO cells were treated with 1 μM FK and 100 nM Cl-IB-MECA in
the absence or presence of different concentrations of the tested
compound (0.01 nM–10 μM). After 15 min incubation, the reaction was
stopped and the intracellular cAMP levels were quantified. Data are
expressed as the percentage of the cAMP intracellular levels with re-
spect to FK, which was set to 100%, and represent the mean SEM of
at least three different experiments. Each experiment was performed
in duplicate.
Fig. 2 Close view of the calculated binding pose for (S)-1 (a) and (R)-1
(b) within a published 3D model of the human A3 AR.18 The ligand and
the receptor are depicted as orange and green sticks, respectively.
H-bonds are depicted as dashed yellow lines.
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subnanomolar affinity for the human A3 AR (0.5 nM) together
with an excellent selectivity profile (no appreciable binding to
human A1, A2A and A2B ARs). These properties make (S)-1 the
most valuable pyrimidine derivative among those investi-
gated so far by us as A3 AR antagonists.
The results described in the present paper will be
exploited for continuing the design and synthesis of novel py-
rimidine derivatives as A3 AR antagonists. Particularly, by tak-
ing 1 as a reference structure, the α-methyl will not be re-
placed by larger groups. Moreover, small substituents on the
phenyl ring will be evaluated in docking simulations for their
capability of establishing favourable interactions with the
surrounding amino acids of the A3 AR binding site.
Fig. 3 Superimposition of the calculated binding poses for (S)-1 (cyan
sticks) and (R)-1 (orange sticks) within a model of the human A3 AR
binding site (green ribbons).
Authorship
The authors have contributed to the work as detailed below.
B. Cosimelli, S. Laneri and A. Sacchi: synthesis. G. Greco
and S. Taliani: design. S. Collina and D. Rossi: HPLC purifi-
cation and analysis. E. Barresi, M. L. Trincavelli and C. Mar-
tini: biological experiments. E. Novellino and S. Cosconati:
docking simulations.
with the Leu264 isobutyl moiety on the opposite side. The
above described interactions seem to provide anchoring points
for the two ligands and allow the placement of the
PhCHIJCH3)O and CH3IJCH2)2S substituents in two different li-
pophilic clefts of the receptor. Specifically, the n-propylthio
chain points towards the inner part of the receptor making van
der Waals contacts with the I186, L91, W243, and L246 side
chains. On the opposite side, the PhCHIJCH3)O group extends
towards the crevice formed by the TM2 and TM3 helices. As
depicted in Fig. 2, it is clear that (S)-1 maximizes the interac-
tions between the terminal phenyl ring and the surrounding
residues (namely Y15, V65, A69, V72, L89, and L90). The same
substituent in (R)-1 still nicely fits within the same cleft. How-
ever, its phenyl ring is less close to the above listed residues,
thus giving rise to less favourable interactions. Our docking
models seem to be consistent with the slightly higher affinity
(3.4-fold) of (S)-1 compared with that of (R)-1.
A close inspection of Fig. 2 reveals that the α-methyl group
of both ligands does not fit into any specific hydrophobic
clefts of the receptor binding site. According to our theoreti-
cal models, the α-methyl group of (S)-1 and (R)-1 acts by
“freezing” the two enantiomers in conformations where the
phenyl and the methyl groups point towards opposite direc-
tions (see Fig. 3), with each conformation capable of optimiz-
ing the favourable interactions that the PhCHIJCH3)O moiety
within the receptor cavity is engaged in.
Conflicts of interest
The authors declare no competing interest.
Acknowledgements
The authors thank Dr. Rita Nasti for support of analytical
work.
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