Navigating in chromone chemical space: Discovery of novel and distinct A3 adenosine receptor ligands

Article abstract of DOI:10.1039/c5ra14988f

One of the major hurdles in the development of safe and effective drugs targeting G-protein coupled receptors (GPCRs) is finding ligands that are highly selective for a specific receptor subtype. The search for novel compounds with therapeutic value by targeting the A₃ adenosine receptor (A₃AR) is still in its early stages. The increasing knowledge about the biological, physiological and pathological role of the A₃AR subtype was accompanied by the design and development of the A₃AR ligands, but the particular role of A₃AR agonists and antagonists is still an open issue. Among the large variety of chemical classes screened towards ARs flavonoids have been indicated as remarkable A₃AR antagonists. However, the search of A₃AR ligands based on this framework seems to be discontinued. In this context, our research group focused its investigation into the discovery and development of novel, potent and selective AR ligands based on the chemical core of flavonoids, the chromone scaffold. The ongoing research has shown that chromone-2-phenylcarboxamide derivatives display a remarkable preference for hA₃AR. In this work we report stimulating results, supported by A_2A/A₃ molecular docking simulations and structure-affinity-relationship (SAR) studies by which N-(4,5-methylthiazol-2-yl)-4-oxo-4H-chromene-2-carboxamide (compound 31) emerged as the most potent and selective compound, displaying an hA₃K_i of 167 nM and a selectivity ratio of 590 vs. the hA₁ and 480 vs. the hA_2AAR subtypes. The chromone-based ligand was obtained by a simple synthetic approach and will enter in a lead optimization program to enhance its potency and drug-like properties.

Full text of DOI:10.1039/c5ra14988f

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Navigating in chromone chemical space: discovery of novel and
distinct A3 adenosine receptor ligands.
F. Cagide,a A. Gaspar,a J. Reis,a D. Chavarria,a S. Vilar,b,c* G. Hripcsak,c E. Uriarte,b S. Kachler,d K.
Klotz,d F. Borgesa *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
One of the major hurdles in the development of safe and effective drugs targeting G-protein coupled receptors (GPCRs) is
finding ligands that are highly selective for a specific receptor subtype. The search for novel compounds with therapeutic
value by targeting the A3 adenosine receptor (A3AR) is still in its early stages. The increasing knowledge about the
biological, physiological and pathological role of A3AR subtype was escorted by the design and development of the A3AR
ligands, but the particular role of A3AR agonists and antagonists is still an open issue.
www.rsc.org/
Among the large variety of chemical classes screened towards ARs flavonoids have been indicated as remarkable A3AR
antagonists. However, the search of A3AR ligands based on this framework seems to be discontinued. In this context, our
research group focused its investigation into the discovery and development of novel, potent and selective ARs ligands
based on the chemical core of flavonoids, the chromone scaffold. The ongoing research has shown that chromone-2-
phenylcarboxamide derivatives display a remarkable tendency for A3AR. In this work we report stimulating results,
supported by A2A/A3 molecular docking simulations and structure-affinity-relationship (SAR) studies by which N-(4,5-
methylthiazol-2-yl)-4-oxo-4H-chromene-2-carboxamide (compound 31) emerged as the most potent and selective
compound, displaying an hA3 Ki of 167 nM and a selectivity ratio of 590 vs. the A1 and 480 vs. the hA2AAR subtypes. The
chromone-based ligand was obtained by a simple synthetic approach and will enter in a lead optimization program to
enhance its potency and drug-like properties.
extracellular adenosine allowed the connection between the
cellular signals promoted by ARs and several types of
Introduction
pathologies,
namely
neurological,
cardiovascular,
Adenosine is a biomolecule that plays a well-known biological
role as nucleic acid building block and as a component of the
cellular ATP energy system. Outside of the cell, adenosine can
act as a signalling molecule by interacting with four distinct
adenosine receptors (ARs) — designated as A1, A2A, A2B and
A3.1,2 The ARs are G protein-coupled receptors (GPCRs) that
modulate adenylate cyclase activity. A1 and A3 receptor
subtypes couple preferentially to Gi protein and thereby inhibit
adenylyl cyclase leading to a reduced production of cyclic AMP
(cAMP) while A2A and A2B subtypes stimulate the production of
cAMP by coupling to Gs proteins.3 The extensive research
dedicated to the physiological and pathological role of
inflammatory diseases and cancer.2,4 Nowadays, all four
adenosine receptors are recognized as valid targets for drug
discovery and development programs.5
Adenosine plays a crucial role in the cell progression pathway,
either during apoptosis or during cytostatic state. It has been
postulated that high levels of extracellular adenosine can have
a profound impact on the growth of tumour masses.6 Several
lines of evidence show that ARs are potential drug targets for
cancer treatment, but it seems that the A2A and A3 subtypes
are the most promising candidates.7 In fact, human A3AR are
overexpressed in tumor cells including leukemia, lymphoma,
astrocytoma and melanoma tumor cells, and are in turn
considered as a target for cancer therapy.8 The receptor is also
overexpressed in inflammatory cells being a putative target for
the treatment of inflammatory disorders, such as rheumatoid
arthritis (RA).8,9 Moreover, A3AR mediated neuroprotective
effects have been demonstrated although further studies are
still needed.8,9
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The increasing knowledge about the physiological and
pathological role of A3AR subtype was escorted by the design
and development of A3AR ligands. From these research
projects, many chemical structures successfully emerged as
potent A3AR agonists (Figure 1)8, 10-12 and antagonists (Figure
2).8,10 Paradoxically, both agonists and antagonists showed
encouraging results namely in anticancer therapy.13-19
However, despite the intensive efforts in medicinal chemistry
to design and synthesize selective A3AR agonists and
antagonists (Figures 2 and 3) from a diversity of chemical
classes few of them have reached the clinical phase.8 Central
drawbacks found for the lack of success include side effects,
which are related to the ubiquity of the receptors, low
absorption, short half-life and toxicity.8
Although benzopyran is considered a privileged structure, few
studies were addressed towards its application in the
discovery of new ARs ligands. In this context, coumarin (2H-
21
chromen-2-one)20,
and flavonoid (2-phenyl-4H-chromen-4-
one)22-24 cores were reported as motivating scaffolds. The
chemical optimization of A3ARs ligands based on a flavonoid
scaffold was reported by Karton et al23 in a study that resulted
in two selective A3AR ligands, MRS 1088 and MRS 1067 with Ki
values of 741 nM and 561 nM, respectively (Figure 3). Yet,
albeit interesting data has been acquired the compounds’ low
aqueous solubility, among other explanations, may be one of
the major problems that hampered progress of the drug
discovery project. To the best of our knowledge, the search for
new and more potent A3AR ligands based on the flavonoid
core was discontinued.
features, along with the structural resemblance with
flavonoids, inspired our group to initiate a medicinal chemistry
program based on the chromone core in order to study its
27
importance in the development of novel AR ligands.26,
Preceding research performed by our group has shown that
chromone-2-phenylcarboxamide is a valid scaffold for the
design of novel A3AR ligands. Therefore, the present study was
accomplished to acquire new data to validate the significance
of the chromone-2-phenylcarboxamide framework. The study
was guided by structure–affinity-relationships (SAR) performed
Chromones are a group of naturally occurring compounds that
are ubiquitous in nature.25 Chemically they are oxygen-
containing heterocyclic compounds with a benzoannelated γ-
pyrone ring (benzo-γ-pyrone [(4H)-1-benzopyran-4- one])
having a great potential for chemical decoration allied with a
broad spectrum of pharmacological activities.25 These
with
a
new library of chromone compounds and
complemented by molecular modeling studies.
Results and discussion
Chemistry
The novel chromone phenylcarboxamides were attained
following synthetic strategies (Scheme 1) that encompassed a
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one-pot condensation between a chromone carboxylic acid
and aryl or heteroaryl amine. The desired compounds were
obtained in moderate to high yields. Strategy 1-A involved the
activation of chromone-2-carboxylic
carboxylic acids by
yloxy)tripyrrolidinophosphonium
(
1
)
or chromone-3-
(benzotriazol-1-
(2)
hexafluorophosphate
(PyBOP) in the presence of N,N-diisopropylethylamine (DIPEA).
After the subsequent addition of the suitable amine
compounds 5-12, 15-18, 21-22, 26-31 and 35 (Tables
were attained. Some chromone-2-carboxamides (19, 20, 23,
25, 32, Table ) were obtained taking advantage of a
microwave-assisted chemistry strategy 1-B Briefly, the
chromone-2-carboxylic acid ( ) was activated by reaction with
1 and 2)
1
.
1
phosphorus oxychloride (POCl3) with formation of the
corresponding acyl chloride, followed by a microwave-assisted
importance of the phenylcarboxamide moiety as a key feature
for receptor binding. Moreover, the location of the amide
function at C-2 or C-3 position of the benzopyran nucleus and
amidation reaction that occurs between the obtained acyl
29
chloride and the corresponding amine.28,
Additionally,
were
the type of amide, preferentially of aromatic type (
2, Table ), has been found to modulate the affinity/selectivity
of the compounds.26-27 It has been also postulated that the
presence of electron withdrawing (Scheme 2, Table ) or
donating groups (Scheme 2, Table ) on the aromatic exocyclic
3, Scheme
chromone-3-carboxamides 33 and 34 (table
2)
1
synthetisized by a slightly modification of strategy 1-B due to
the lability of the starting material and the inherent formation
of enamine by-products.30 The reaction of chromone-3-
1
1
carboxylic acid (2) with POCl3 led to the formation of the
ring seems to modulate the affinity and selectivity of
chromone-2-phenylcarboxamide towards ARs subtypes.26-27
New derivatives with carefully chosen substituents were
synthesised in order to acquire more information about the
significance of chromone-2-phenylcarboxamide as putative
corresponding acyl chloride and the amidation reaction took
place at room temperature, without the employment of
microwave heating (Scheme 1-C).
Pharmacology
A3AR ligand (Scheme 1, Table 1). Their influence on affinity and
selectivity for human A1, A2A and A3 ARs was evaluated in
radioligand binding while the interaction with the A2B AR was
analyzed by adenylyl cyclase activity measurement following
The affinity of the new potential antagonists for the human
adenosine receptor subtypes hA1, hA2A, hA3 (expressed in
Chinese hamster ovary (CHO) cells) was determined in
radioligand competition experiments.11, 31-34 In this assay, the
competition with the following agonist radioligands: (i)
[3H]CCPA at hA1 receptors, (ii) [3H]NECA at hA2A and
[3H]HEMADO at hA3 receptors was measured. The data were
expressed as Ki (dissociation constant). The receptor binding
the procedures previously described by Klotz et al. with minor
modifications.11,
31-34
For all the tested compounds no
measurable activity for A2B AR (Ki > 10,000 or 30,000 nM) was
detected and therefore these values were omitted in Table
1.
Previously, it was postulated that the presence of electron
donors or withdrawing groups on the exocyclic aromatic
system can modulate the affinity and selectivity of
chromones.26 Therefore, it was decided to synthesise a new
chromone-2-phenylcarboxamide series that incorporates
diverse types of substituents located at different positions of
the exocyclic aromatic nucleus. These structural modifications,
namely in para position, enabled the development of an
extended SAR study. In accordance with the previous data, it
was found that the introduction of different electron
withdrawing substituents with dissimilar relative strengths
from weak (-F, -Br, -I, compounds 5-7), to moderate (-COCH3, -
affinities of the synthesised compounds (
Tables 1 and
3-35) are reported in
2.
Structure-affinity relationship studies
Foregoing research of our group allowed acquiring evidence
that validate chromone as a privileged structure for the design
of adenosine
receptor
ligands and
chromone-2-
phenylcarboxamide (Scheme
2
) as a new lead compound for
the development of A3AR ligands.26,27 The structure-activity
relationship (SAR) studies performed so far revealed the
COOC2H5, compounds 8 and 9) and strong (-CN, compound 10)
results in all cases in compounds with no measurable affinity
for all AR subtypes. Furthermore, a lack of affinity is also
observed for a Cl-substituent in para (compound
4), ortho
(compound 11) and meta (compound 12) position of the
exoxyclic phenyl ring. Thus, the binding of chromone-2-
phenylcarboxamides to AR is strongly influenced by electronic
properties of the substituents and that any type of electron
withdrawing groups located at any position of the exocyclic
aromatic substituent is not tolerated.
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The results obtained from the introduction of electron reliability of the SAR studies, and taking into account the
donating substituents in the exocyclic ring were not as importance of the aromaticity of the carboxamide moiety for
straightforward as it was for the electron withdrawing groups. the affinity towards A3AR, a series of heterocyclic carboxamide
Preliminary data obtained in previous studies emphasizes the derivatives were synthesised and evaluated towards all ARs
effect of electron donating groups located in para position of subtypes (compounds 27
the exocyclic phenyl ring of the chromone-2- heterocyclic ring, directly linked to the NH of the carboxamide,
phenylcarboxamide as it appear to modulate the resulted in active chromone-2-carboxamides with moderate to
affinity/selectivity for the A3AR.26 From these studies one can good binding affinities for human A1, A2A and A3 ARs. However,
highlight the data showing the influence of a p-methyl (Ki= the replacement of the phenyl (compound , Ki= 14,200 nM)
15,800 nM, compound 13) and a p-methoxyl substituent (Ki = by an isosteric pyridine ring (compound 27, Ki= 10,700 nM) or
9,580 nM compound 14) on A3AR affinity (Table
).26 Hence, thiazol (compound 29, Ki= 11,900 nM) gave rise to compounds
-32). In general, the introduction of a
(3)
3
1
guided by this information it was decided to study the with similar affinity towards A3ARs but a noticeable loss of
influence on affinity of the lengthening of the saturated carbon selectivity was observed. Moreover, the presence of a CH2
side chain, from one (-CH3, compound 13), two (-C2H5, spacer between the carboxamide and the exoheterocyclic ring
compound 15) and four (-C4H9, compound 16) carbon atoms. A (compound 28) led to a complete loss of affinity towards A3AR,
complete loss of affinity towards all ARs subtypes was although A1 (Ki=12,000 nM) and A2A receptor (Ki=18,500 nM)
observed. These results suggest that the affinity of the binding affinities were detected. Inspiring results were
chromone-2-phenylcarboxamides derivatives to ARs may be obtained with the chromone-2-carboxamides bearing
a
circumscribed by steric hindrance factors. Then, to assess the substituted thiazolyl group (compounds 30-32). The
influence of the position of the electron donating substituent introduction of a methyl substituent at position 5 of the
on the aromatic exocyclic nucleus, several positional isomers heterocyclic ring (compound 30, Ki= 1,310 nM) gave rise to
were synthesized. A decrease or absence of affinity was compounds about six- and eightfold selective for A3 versus A1
observed for substituents located either in ortho (CH3, (Ki= 8,180 nM) and A2A (Ki= 10,400 nM) ARs, respectively. The
compound 17; OCH3, compound 19) or meta (CH3, compound introduction of an additional substituent at position 4 of the
18; OCH3, compound 20) positions. Noteworthy data was thiazol moiety (compounds 31 and 32) led to even more
obtained by the isosteric replacement of the methoxyl group selective and potent chromone-2-carboxamide derivatives. In
(compound 14) by a thiomethyl (compound 21) as it results in fact, compound 31 was found to be the most potent and
a compound with no affinity to all ARs. The effect of the selective compound of all the chromone-2-carboxamide series
presence of two electron donating substituents (-CH3 and/or - tested so far, displaying a Ki= 167 nM for A3AR and selectivity
OCH3) located in diverse positions (vicinal or meta position) of ratios of 590 and 480 compared to A1 and A2A ARs,
the exocyclic phenyl ring (compounds 22-26) on ARs affinity respectively. The loss in potency observed with compound 32
was also inspected. A decrease (2,3-diCH3, compound 23 and (A3 Ki= 4,780 nM) can be explained with the introduction of an
3,4-diOCH3, compound 24) or no measurable affinity (3,4- electron withdrawing group (-COOEt) in the mentioned
diCH3, compound 22) for the A3AR subtype was observed. position.
Interestingly, the introduction of a para methoxyl and an ortho The compelling results associated to the chromone-2-(thiazol-
methyl substituents (2-CH3-4-OCH3, compound 25) gave rise to 2-yl)carboxamides prompted to the synthesize of their
an inactive compound. The same tendency has been positional C-3 isomers in order to corroborate the current SAR
previously observed with compound 24, with two vicinal studies. The results for the binding affinity obtained for the
methoxy groups,26 and compound 26 that possesses
a
chromone-3-(thiazol-2-yl)carboxamides
summarized in Table
(
33
-35)
are
methylenedioxy substituent on the exocyclic phenyl ring.
2.
Our results reported here corroborate the preliminary SAR In analogy to the C-2 isomers no measurable activity for A2B AR
data observed in the previous studies.26 In order to check the (Ki > 100,000 or 10,000 nM) was detected for the C-3
33
34
> 30,000
> 10,000
> 30,000
> 10,000
32,400 (25,100-41,800)
>10,000
>0.9
__
>0.9
__
35
> 10,000
> 10,000
10,000
__
__
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carboxamides. From the analysed data it is possible to
conclude that the position of the (thiazol-2-yl)carboxamide
moiety at the chromone core is crucial for the observed
affinity. In fact a decrease or a lack of affinity for all ARs was
observed for compounds 33
,
34 and 35 that are positional
isomers of compounds 30
, 31, and 32, respectively.
Molecular docking
Molecular docking simulations on hA2A and hA3 receptors were
performed to study the binding modes of the most active
compounds in the series. To carry out this step, the crystal
structure 3EML (PDB code)35 for the hA2A and a homology
model for the hA3 ARs were used (see Experimental Section for
more details).21 The ligands were docked using Glide in the
Schrodinger package.36 For validation process the co-
crystallized ligands ZM241385 and T4E in the structures 3EML
35
(including the role of water in the pocket) and 3UZC37 were
docked to the hA2A protein. A RMSD (root mean square
deviation) was obtained between the theoretical poses and
the co-crystallized conformations of 0.69 and 1.92 for the
ZM241385 and the triazine derivative T4E, respectively.
The docking calculations of chromone compounds showed a
high degree of variability regarding possible binding modes in
the pocket, in particular in the case of the hA3 homology
model. Selection of the described binding modes was carried
out taking into account the energetic parameter Emodel and the
number of similar poses yielded by the simulations. In both
hA2A and hA3 the active compounds with a thiazole ring
(compounds 29 and 30) shared a similar binding mode inside
the AR subtype receptors. For instance compound 30 oriented
the thiazole ring towards the bottom of the cavity whereas the
chromone ring is placed closer to the extracellular
environment in both receptors. The described binding mode
presents some similarity to the co-crystallized antagonist
ZM241385 in the hA2A as it orientates the furan ring towards
the bottom of the pocket in the same area as the thiazole ring
of compound 30 (see Figure 4a). In the same manner as the co-
crystallized ligand the compound established hydrogen bonds
with the residue Asn253 of the hA2A (see Figure 4b). Moreover,
the hydrophobic/hydrophilic surface generated by the protein
was also plotted. The surface revealed favored hydrophobic
and hydrophilic areas for ligand interaction that can help to
understand the pose of the compounds inside the protein. In
fact, for compounds 29 and 30 a good accommodation for the
thiazole ring was attained (see Figure 4c) whereas the
introduction of a second 4-methyl substituent in the thiazole
(compound 31) lead to a loss of its fitting in the hydrophobic
area. The loss of this hydrophobic interaction can cause the
disruption of the binding mode being an important factor to
explain the lack of affinity of compound 31 for the hA2AAR
(Ki>80,000 nM) compared to the derivatives 29 and 30
(Ki=15,100 nM and Ki=10,400 nM, respectively). Binding mode
of compound 30 was also inspected by docking studies with
water molecules present in the pocket of the crystallized
structure 3EML. Compound 30 binding mode was similar as
the one previously described. Thiazole ring pose is oriented
towards the bottom of the cavity and established hydrogen
bonds with residue Asn253 and water molecules (see Figure
4d).
Thiazole chromone derivatives were also inspected in docking
to the hA3 AR. The pose obtained for compound 31, the most
active compound in the series, is very similar to that obtained
in the binding mode studies already described for the hA2A (see
Figure 5a). The analysis of the binding mode showed that the
thiazole ring plays a key role in the anchoring of the amide
moiety to the residue Asn250 through hydrogen bond
interactions. Moreover, the oxygen of the carbonyl group in
the chromone nucleus played an important role interacting
with residue Gln167 of the second extracellular loop (EL2). In
the case of the hA2A, the residue Gln167 is not present as the
protein contains a Leu in this position and so no hydrogen
bond with the ligand is detected. This residue can be an
operative feature in the ligand binding that can explain the
enhanced affinity shown by the thiazole derivatives in the hA3.
On the other hand, hydrophobic/hydrophilic surfaces in the
hA3 pocket showed important differences compared to the
hA2A in the area occupied by the thiazole ring. In the case of
the hA3, the hydrophobic surface is larger thus allowing
hydrophobic substitutions at position 4 of the thiazole (see
Figure 5b). This fact could explain the enhancement of hA3
activity of compound 31 (Ki=167 nM) with regard to derivatives
29 and 30 (Ki=11,900 nM and Ki=1,310 nM respectively).
Moreover, the distinct hydrophobic distribution detected in
both proteins (hA2A and hA3) can elucidate the different
adenosine selectivity of compound 31
.
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The hA3 also presents a favored hydrophobic surface close to
the extracellular environment. As it was previously reported,38
there are some differences in the extracellular loops of the
hA2A and hA3 that could determine not only the way how
ligands bind to the protein but also modulate the entrance of
ligands to the interior of the binding cavity. The different
nature of some extracellular residues in both proteins, such as
the hydrophobic Val169 in the hA3 and the ionic Glu169 in the
31 and the hA3 were determined: Phe168, Asn250, Leu246,
Ile268 and Gln167. The higher interaction of compound 31
with the key residue Asn250 can also validate its superior hA3
activity. It is noteworthy that although hA3 possesses high
identity with the hA2A there are also differences in some
residues of the binding pocket which can condition the binding
of the different ligands.
The position of the carboxamide in the chromone ring was also
found to be important for A3 adenosine receptor affinity. In
fact, compounds with the carboxamide function at position 3,
such as compound 34, showed a drastic loss of hA3 activity.
Docking calculations in hA3 regarding compound 34
demonstrated a hypothetical binding mode with the chromone
ring in a similar position as the one described for compound
31. However, compound 34 oriented the carboxamide
substituent towards the extracellular environment. The
different binding mode could be responsible for the loss of hA3
activity.
hA2A, could influence the access and accommodation of the
ligands. Introduction of a second methyl in the thiazole
fragment contributed to an increase of hA3 activity as the
receptor present a deeper hydrophobic surface.
In addition, molecular modeling studies were performed with
the chromone-2-phenylcarboxamide with
a
methoxyl
Moreover, the contribution to the interaction energy of the
different residues placed in a distance of 4 Å from the ligand in
the hA2A and hA3 was calculated. The interaction energy
(Figure 6) was calculated as the sum of three scores: Coulomb,
van der Waals and hydrogen bonding. Compound 30 inside the
hA2A interacts preferably with residues Phe168, Asn253,
Leu249, Ile274 and Met177. Although compound 31 presents a
similar interaction outline a lower interaction with residue
His250 was noticed. The important role of some residues in
ligand binding, such as Phe168, Asn253, Leu249 or Met177
was previously confirmed in mutagenesis studies that
examined the effect of mutating residues in hA2A activity.38 On
the other hand, and using the same approach, the most
substituent located in para position of the exocyclic aromatic
ring (14). It was concluded that this type of chromone
presented a different binding mode in the hA3AR (see Figure
5c). Compound 14 oriented the chromone scaffold towards
the bottom of the cavity and the para-methoxyphenyl moiety
towards the extracellular region through the interaction of
hydrogen bonds with residues Asn250 and Gln167. Similar
binding
modes
for
this
type
of
chromone-2-
phenylcarboxamide derivatives were previously reported by
our research group.26So, aromatic heterocyclic systems are
considered to be positive bioisosteric replacements to improve
hA3AR affinity of this type of ligands.
important residues in the interaction between compounds 30
-
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28
29
30
31
32
33
34
35
-7.54
-7.46
-7.72
-7.50
-6.83
-7.73
-6.57
-7.06
-8.09
-6.44
-7.88
-7.85
-8.11
-7.89
-8.17
-7.49
-7.88
-7.94
-8.31
-8.38
-6.10
-7.65
-8.13
-6.25
-7.28
-6.50
-7.14
-7.21
-7.15
-7.34
-7.58
-7.21
-8.29
-8.11
-8.45
-8.32
-8.44
-7.55
-7.14
-8.31
-7.91
-6.60
-6.29
-7.08
-7.76
-7.37
-7.87
-7.96
From the overall studies one can conclude that the
hydrophobic area in the extracellular environment due to the
presence of hydrophobic residues, such as Val169 and Leu264,
can be considered a significant feature to be accomplished
along compound 31 optimization subsequent step. The change
of the type of heterocycle is considered to be an important
approach to obtain additional information, improve the affinity
and validate the docking studies. Also the presence of other
SP (standard precision scoring function), XP (extra-precision scoring function, kcal/mol).
For AUROC calculation, compounds 27-30 were actives in the hA2A, and compounds 14,
30, 31, 32 and 35 were actives in the hA3 (Ki≤10,000 nM). Calculations with and without
type of electron donator substituents, which must be oriented water molecules in the hA2A pocket were performed.
toward the extracellular area, must be explored.
Scoring function energies for each compound docked to the hA2A
Conclusions
and hA3 are shown in Table 3. From the data one can conclude that
molecular docking showed limited ability to discriminate active
from non-active compounds, even though the results are variable
depending on the scoring function (SP or XP) and the adenosine
The search for novel compounds with therapeutic value by
targeting A3 ARs is still in its infancy and additional research in
the field is still needed. The role of A3AR agonists and
subtype. SP (standard precision) scoring function showed better antagonists is still an open issue and the development of new
and superior agonists/antagonists that can reach clinical trials
remains an emergent topic. Previous research performed by
our group has shown that chromone-2-phenylcarboxamide is a
valid scaffold for tracking novel ligands with affinity for A3AR.
Thus, the study presented here provides new insights into the
discriminatory power than the XP (extra-precision) scoring. The area
under the ROC curves (AUROC) was also calculated to evaluate the
discriminatory ability in the series. SP scoring showed AUROCs of
0.77 and 0.64 in the hA2A (including water or not in the pocket). The
most hA2A active compound (30) in the series is identified by the SP
models in position 2 and 8, respectively. Docking in hA3 showed
more limitations to differentiate between active and non-active
compounds. However, hA3 docking with SP function showed some
ability to capture the most active compounds in the series in top
positions (positions 9, 10, 13, 15 and 16 for compounds 30, 32, 14,
31 and 35 respectively). AUROC is 0.66 considering as actives the
compounds with Ki≤10,000 nM. Docking studies showed some
limitations from the energetic point of view in the hA2A crystal
structure and the hA3 homology model.
SAR
regarding
the
chromone-2-phenylcarboxamide
framework. This input, supported by A2A/A3 molecular docking
simulations and structure-affinity-relationship (SAR) studies,
lead to the identification of the crucial role for A3AR affinity of
a heteroamide substituent located at C-2 of the pyrone ring.
The most remarkable chromone-2-carboxamides with affinity
towards A3ARs were compounds 30, 31 and 32 with Ki values
of 1,310 nM, 167 nM and 4,780 nM, respectively. Compound
31, displaying an A3 Ki of 167 nM and a selectivity ratio of 590
vs. the A1 and 480 vs. the A2AAR subtypes, stood out as the
best chromone derivative tested so far, showing an enhanced
A3AR affinity and selectivity compared to the flavonoids
presently known. Moreover, compound 31 present appealing
drug-like properties (e.g. clogP 2.44 and molecular polar
surface area of 72.20) and no violations of Lipinski rule.
hA2A
hA2A
hA3
SP (no
water)
0.64
XP (no
water)
0.47
SP
(water)
0.77
XP
(water)
0.66
SP (no
water)
0.66
XP (no
water)
0.36
AUROC
Comp.
SP (no
water)
-7.80
-6.61
-7.60
-6.81
-7.16
-7.80
-6.32
-7.53
-8.37
-7.23
-7.69
-7.83
-7.58
-5.94
-6.84
-7.43
-5.82
-7.72
-7.64
-8.31
-6.89
-6.76
-7.11
-7.13
-7.57
XP (no
water)
-8.33
-8.62
-7.72
-7.82
-7.56
-8.60
-9.07
-7.53
-8.30
-8.66
-8.28
-8.37
-7.86
-9.45
-3.70
-8.58
-8.71
-8.60
-7.32
-8.70
-8.73
-5.43
-8.86
-6.84
-9.77
SP
(water)
-7.27
-7.56
-7.75
-7.09
-6.76
-6.10
-6.77
-6.51
-5.46
-7.68
-7.49
-5.13
-6.74
-5.47
-7.12
-7.76
-7.62
-6.14
-6.38
-8.19
-7.18
-5.94
-5.62
-6.40
-6.34
XP
(water)
-6.95
-6.79
-6.28
-6.39
-5.98
-5.08
-6.15
-6.40
-5.23
-7.74
-6.33
-5.95
-6.67
-5.24
-6.85
-7.64
-6.87
-5.11
-5.84
-6.62
-6.26
-6.22
-5.36
-7.21
-6.52
SP (no
water)
-7.78
-8.48
-7.93
-8.30
-7.28
-9.01
-8.68
-8.62
-7.54
-7.93
-7.65
-8.35
-8.30
-8.66
-7.97
-7.82
-8.03
-8.44
-7.86
-8.40
-8.29
-8.62
-8.71
-8.32
-8.57
XP (no
water)
-7.06
-8.22
-7.56
-7.87
-7.46
-8.19
-7.47
-7.42
-9.49
-7.69
-8.05
-8.10
-7.77
-8.40
-7.49
-8.01
-7.98
-7.94
-7.32
-8.48
-8.13
-8.77
-9.04
-7.82
-6.80
3
In addition, the present study supports the belief that
chromone due to its synthetic accessibility and decoration
4
5
6
capability, is a privileged structure for the design and
7
development of libraries in the discovery of novel and
innovative ARs ligands. Finally, the receptor-driven molecular
modeling studies have provided valuable information on the
molecular interactions responsible for selective high affinity
binding to the A3 adenosine receptor of the most promising
chromones. In this respect the data are instrumental for future
optimization of potency and drug-like properties of chromone-
2-(hetero)carboxamides.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Experimental section
Chemistry
Chromone-2-carboxylic
acid,
benzotriazol-1-
yloxy)tripyrrolidinophosphonium
hexafluorophosphate
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N-phenyl-4-oxo-4H-chromene-2-carboxamide
(
3):
the
(PyBOP),
N,N-diisopropylethylamine
(DIPEA),
synthetic procedure and structural elucidation data was
described elsewhere.44
dimethylformamide (DMF), phosphoryl trichloride (POCl3),
arylamines and heteroamines were purchased from Sigma-
Aldrich Química S.A. (Sintra, Portugal). All other reagents and
solvents were pro analysis grade and were acquired from
Merck (Lisbon, Portugal), Panreac (Lisbon, Portugal) and Sigma
Aldrich (Sintra, Portugal), used without additional purification.
Thin-layer chromatography (TLC) was carried out on pre-
coated silica gel 60 F254 (Merck) with layer thickness of 0.2
mm. The spots were visualized under UV detection (254 and
366 nm) and iodine vapour. Normal-phase column
chromatography was performed using silica gel 60 0.2-0.5 or
0.040-0.063 mm (Merck).
N-(4-chlorophenyl)-4-oxo-4H-chromene-2-carboxamide
(4):
the synthetic procedure and structural elucidation data was
described elsewhere.45
N-(4-Fluorphenyl)-4-oxo-4H-chromene-2-carboxamide
(5):
Yield: 75%. 1H NMR (DMSO-D6): δ = 6.98 (1H, s, H(3)), 7.28
(2H, dd, J = 8.9, H(3’), H(5’)), 7.57 (1H, ddd, J = 8.0, 7.0, 1.0,
H(6)), 7.81-7.85 (3H, m, H(8), H(2’), H(6’)), 7.94 (1H, ddd, J =
1.6, 7.0, 8.6, H(7)), 8.09 (1H, dd, J = 1.6, 8.0, H(5)), 10.81 (1H, s,
CONH)). 13C NMR (DMSO-D6): δ = 112.5 (C(3)), 116.9 (d, J =
22.5, C(3’), C(5’)), 120.4 (C(8)), 124.5 (d, J = 8.2, C(2’), C(6’)) ,
125.1 (C(4a)), 126.3 (C(6)), 127.5 (C(5)), 135.2 (C(7)), 136.5
(C(1’)), 156.5 (C(8a)), 157.0 (CONH), 159.1 (C(2)), 160.4 (d, J =
Following the workup and after extraction, the organic phases
were dried over Na2SO4. Solutions were decolorized with
activated charcoal, when necessary. Solvents were evaporated
in a Buchi Rotavapor.
+
•
241.9, C(4’)) 178.7 (C(4)). EM/IE m/z: 284 (M+1, 21), 283 (M ,
100), 282 (83), 254 (29), 145 (23), 89 (58).
The purity of the final products (>97% purity) was verified by
high-performance liquid chromatography (HPLC) equipped
with a UV detector. Chromatograms were obtained in an
HPLC/DAD system, a Jasco instrument (pumps model 880-PU
N-(4-Bromophenyl)-4-oxo-4H-chromene-2-carboxamide
(6):
the synthetic procedure and structural elucidation data was
described elsewhere.28
and solvent mixing model 880-30, Tokyo, Japan), equipped N-(4-Iodophenyl)-4-oxo-4H-chromene-2-carboxamide (
7) Yield:
95% 1H NMR (DMSO-D6): δ = 6.97 (1H, s, H (3)), 7.56 (1H, ddd,
J = 1.0, 7.1, 8.0, H(6)), 7.65 (2H, d, J = 8.8, H(3’), H(5’)), 7.77
(2H, d, J = 8.8, H(2’), H(6’)), 7.84 (1H, d, J = 8.5, H(8)), 7.93 (1H,
ddd, J = 1.6, 7.0, 8.6, H(7)), 8.08 (1H, dd, J = 1.4, 8.0, H(5)),
10.82 (1H, s, NH). 13C NMR (DMSO-D6): δ = 90.5 (C(4’)), 112.6
(C(3)), 120.4 (C(8)), 124.5 (C(2’), C(6’)), 124.6 (C(4a)), 126.3
(C(6)), 127.5 (C(5)), 136.5 (C(7)), 138.8 (C(1’)), 138.9 (C(3’),
C(5’)), 156.5 (C(8a)), 156.8 (C(2)), 159.2 (NH), 178.6 (C(4)).
with a commercially prepacked RP-18 analytical column (250
mm x 4.6 mm, 5 m, Macherey-Nagel, Duren, Germany), and
UV detection (Jasco model 875-UV) at the maximum
wavelength of 254 nm. The mobile phase consisted of a
methanol/water or acetonitrile/water (gradient mode, room
temperature) at a flow rate of 1 mL/min. The chromatographic
data was processed in a Compaq computer, fitted with CSW
1.7 software (DataApex, Czech Republic).
Apparatus. The main paragraph text 1H NMR and 13C NMR data
+
•
MS/EI m/z: 392 (22), 391 (M , 100), 390 (47), 362 (16), 145
were acquired, at room temperature, on a Brüker AMX 400
spectrometer operating at 400.15 and 101.0 MHz,
respectively. Chemical shifts are expressed in (ppm) values
relative to tetramethylsilane (TMS) as internal reference;
coupling constants (J) are given in Hz. Electron impact mass
(13), 89 (45), 86 (12), 84 (18), 70 (10).
N-(4-Acetylphenyl)-4-oxo-4H-chromene-2-carboxamide (8):
1H NMR (CDCl3): δ = 2.66 (3H, s, CH3), 7.38 (1H, s, H(3)), 7.55
(1H, app t, J = 7.8, H(6)), 7.78 (1H, d, J = 8.6, H (8)), 7.85 (1H,
ddd, J = 1.5, 7.8, 8.0, H(7)), 7.95 (2H, d, J = 8.7, H(3’), H(5’)),
8.06 (2H, d, J = 8.7, H(2’), H(6’)), 8.26 (1H, dd, J = 7.8, 1.5, H(5)),
9.86 (1H, s, CONH)). 13C NMR (CDCl3): δ = 26.4 (CH3), 112.4
(C(3)), 118.4 (C(8)), 120.2 (C(3’), C(5’)), 124.1 (C(4a)), 125.9
(C(5)), 126.3 (C(6)), 129.7 (C(2’), C(6’)) 133.7 (C(1’)), 135.1
(C(7)), 141.3 (C(4’)), 154.9 (C(8a)), 155.5 (C(2)), 157.8 (CONH),
spectra (EI-MS) were carried out on
a VG AutoSpec
instrument; the data are reported as m/z (% of relative
intensity of the most important fragments).
Synthesis. The chromone derivatives were synthesised by two
synthetic approaches previously developed by our group.28,29
Method A: To a solution of the chromone-2-carboxylic acid or
chromone-3-carboxylic (1.1 mmol) in DMF (2.5 mL) at 4 ºC it
was added was added N,N-diethylpropan-2-amine (1.1 mmol)
and a solution of PyBOP (1mmol) in CH2Cl2 (2.5 mL). The
mixture was kept in an ice bath and stirred for half hour. After
this period the amine with the desired (hetero)aromatic
pattern was added and the mixture was allowed to warm up to
room temperature. The reaction was kept under stirring for 4
hours. The working-up of the crude material enclosed a liquid-
liquid extraction CH2Cl2 followed by flash chromatography
(CH2Cl2/MeOH or EtOAc/n-hexane) and final purification by
recrystallization (EtOAc/n-hexane).
+
•
178.7 (C(4)), 197.8 (COCH3). MS/EI m/z: 307 (M , 87), 292
(100), 146 (19), 121 (37), 89 (56).
N-(4-Ethoxycarbonylphenyl)-4-oxo-4H-chromene-2-
carboxamide (9
): Yield: 82 %. 1H NMR (CDCl3): δ = 1.41 (3H, t, J
= 7.1, CH2CH3), 4.39 (2H, d, J = 7.1, CH2CH3), 7.28 (1H, s, H(3)),
7.50 (1H, ddd, J = 1.0, 7.1, 8.0, H(6)), 7.61 (1H, d, J = 8.5, H(8)),
7.79 (1H, ddd, J = 1.6, 7.0, 8.6, H(7)), 7.82 (2H, d, J = 8.8, H(3’),
H(5’)), 8.11 (2H, d, J = 8.8, H(2’)), H(6’)), 8.25 (1H, dd, J = 1.4,
8.0, H(5)), 8.74 (1H, s, CONH). 13C NMR (CDCl3): δ = 14.3
(CH2CH3), 61.1 (CH2CH3), 112.9 (C(3)), 118.0 (C(8)), 119.5 (C(2’),
C(6’)), 123.9 (C(4a)), 124.3 (C(4’)), 126.3 (C(6)), 127.4 (C(5)),
130.9 (C(3’), C(5’)), 134.8 (C(7)), 140.3 (C(1’)), 154.1 (C(8a)),
155.1 (C(2)), 157.1 (CONH), 177.0 (C(4)). EM/IE m/z: 338 (37),
+
•
337 (94), 336 (M , 100), 320 (24), 309 (13), 308 (53), 293 (18),
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ARTICLE
292 (81), 280 (14), 265 (17), 264 (18), 146 (17), 145 (18), 121 N-(4-(Methylthio)phenyl)-4-oxo-4H-chromene-2-carboxamide
(29), 120 (12), 101 (12), 89 (63).
(
21): the synthetic procedure and structural elucidation data
N-(4-Cyanophenyl)-4-oxo-4H-chromene-2-carboxamide
Yield: 71%. 1H NMR (DMSO-D6): δ = 7.01 (1H, s, H(3)), 7.56 N-(3,4-Dimethylphenyl)-4-oxo-4H-chromene-2-carboxamide
(
10): was described elsewhere.40
(1H, ddd, J = 1.2, 7.4, 7.8, H(6)), 7.82 – 7.87 (3H, m, H(3’), H(5’),
(
22): Yield: 75% 1H NMR (DMSO-D6): δ = 2.21 (3H, s, CH3), 2.24
H(8)), 7.93 (1H, ddd, J = 1.6, 7.6, 8.0, H(7)), 7.98 (2H, d, J = 8.8, (3H, s, CH3), 6.95 (1H, s, H(3)), 7.16 (1H, d, J = 8.0, H(5’)), 7.52-
H(2’), H(6’)), 8.08 (1H, dd, J = 1.2, 8.0, H(5)), 11.09 (1H, s, NH). 7.54 (1H, m, H(2’)), 7.56-7.58 (2H, m, H(6’), H(6)), 7.84 (1H, d, J
13C NMR (DMSO-D6): δ = 105.3 (C(4’)), 110.6 (C(3)), 110.7 (CN), = 8.0, H(8)), 7.93 (1H, ddd, J =1.2, 6.8, 8.0, H(7)), 8.08 (1H, dd, J
118.4 (C(2’), C(6’)), 121.0 (C(8)), 123.1 (C(4a)), 124.3 (C(6)), = 1.2, 8.0, H(5)), 10.60 (1H, s, NH). 13C NMR (DMSO-D6): δ =
125.5 (C(5)), 132.5 (C(3’), C(5’)), 134.4 (C(7)), 143.1 (C(1’)), 20.3 (CH3), 21.0 (CH3), 112.3 (C(3)), 119.9 (C(8)), 121.4 (C(2’)),
154.6 (C(8a)), 155.6 (C(2)), 158.2 (NH), 176.7 (C(4)). EM/IE m/z: 123.5 (C(6)), 125.1 (C(4a)), 126.3 (C(6’)), 127.5 (C(5)), 131.0
+
•
(C(3’)), 134.3 (C(4’), C(5’)), 136.4 (C(7)), 137.9 (C(1’)), 156.5
290 (M , 91), 289 (100), 261 (25), 173 (26), 145 (39), 89 (88),
(C(8a)), 157.2 (C(2)), 158.8 (CONH), 178.7 (C(4)). MS/EI m/z:
+
11): 293 (M , 100), 292 (90), 276 (25), 264 (35), 173 (22), 120 (45),
69 (20).
•
N-(2-Chlorophenyl)-4-oxo-4H-chromene-2-carboxamide
(
the synthetic procedure and structural elucidation data was 89 (49), 77 (24).
described elsewhere.39
N-(3,4-Dimethoxyphenyl)-4-oxo-4H-1-benzopyran-2-
N-(3-Chlorophenyl)-4-oxo-4H-chromene-2-carboxamide
(12): carboxamide (24): the synthetic procedure and structural
the synthetic procedure and structural elucidation data was elucidation data was described elsewhere.26
described elsewhere.39
N-(Benzodioxol-5-yl)-4-oxo-4H-chromene-2-carboxamide
(26)
N-(p-Tolyl)-4-oxo-4H-chromene-2-carboxamide
(13):
the Yield: 62%. 1H NMR (DMSO-D6): δ = 6.05 (2H, s, CH2), 6.95 (1H,
synthetic procedure and structural elucidation data was s, H(3)), 6.97 (1H, d, J = 8.4, H(5’)), 7.24 (1H, dd, J = 2.4, 8.4,
described elsewhere.26
H(6’)), 7.43 (1H, d, J = 2.4, H(2’)), 7.57 (1H, ddd, J = 1.0, 7.6, 9.2,
N-(4-methoxyphenyl)-4-oxo-4H-chromene-2-carboxamide
H(6)), 7.84 (1H, dd, J = 0.8, 8.4, H(8)), 7.93 (1H, app dt, J = 1.6,
(
14): the synthetic procedure and structural elucidation data 7.8, H(7)), 8.08 (1H, dd, J = 1.6, 8.0, H(5)), 10.65 (1H, s, NH). 13C
was described elsewhere.26
NMR (DMSO-D6): δ = 100.7 (CH2), 102.4 (C(2’)), 107.5 (C(5’)),
N-(4-Ethylphenyl)-4-oxo-4H-chromene-2-carboxamide
(
15): 110.4 (C(6’)), 113.8 (C(3)), 118.4 (C(8)), 123.1 (C(4a)), 124.4
1
Yield: 67%. H NMR (CDCl3): δ = 1.24 (3H, t, J = 7.6, CH3), 2.65 (C(6)), 125.5 (C(5)), 131.1 (C(1’)), 134.5 (C(7)), 143.7 (C(4’)),
(2H, J = 7.6, CH2), 7.23 (2H, d, J = 8.5, H(3’), H(5’)), 7.25 (1H, s, 146.5 (C(3’)), 154.6 (C(8a)), 155.1 (C(2)), 156.8 (NH), 176.7
+
•
H(3)), 7.47 (1H, ddd, J = 1.0, 7.2, 8.0, H(6)), 7.57-7.65 (3H, m,
H(8), H(2’), H(6’)), 7.76 (1H, ddd, J = 1.5, 7.2, 8.7, H(7)), 8.23
(1H, dd, J = 1.5, 8.0, H(5)), 8.55 (1H, s, NH). 13C NMR (CDCl3): δ
= 15.5 (CH3), 28.4 (CH2), 112.3 (C(3)), 118.1 (C(8)), 120.6 (C(2’),
C(6’)) , 124.4 (C(4a)), 126.1 (C(6)), 126.2 (C(5)), 128.6 (C(3’),
C(5’)), 134.0 (C(4’)), 134.7 (C(7)), 142.0 (C(1’)), 154.7 (C(8a)),
(C(4)). MS/EI m/z: 310 (M+1, 25), 309 (M , 100), 292 (17), 280
(18), 189 (30), 136 (65), 89 (33).
N-(Pyridin-2-yl)-4-oxo-4H-chromene-2-carboxamide (27): Yield:
31%. 1H NMR (CDCl3): δ = 7.19 (1H, ddd, J = 0.9, 5.0, 7.4, H(4’)),
7.30 (1H, s, H(3)), 7.50 (1H, ddd, J = 1.0, 7.2, 8.1, H(6)), 7.63
(1H, dd, J = 0.6, 8.5, H(6’)), 7.89-7.72 (2H, m, H(5’),H(8)), 8.25
+
•
155.2 (C(2)), 156.8 (CONH), 178.0 (C(4)). MS/EI m/z: 293 (M , (1H, dd, J = 1.4, 8.0, H(3’)), 8.45-8.32 (2H, m, H(5), H(7)), 9.31
(1H, s, NH). 13C NMR (CDCl3): δ = 113.0 (C(3)), 114.8 (C(6’)),
100), 278 (20), 264 (20), 120 (31), 107 (23), 89 (99), 77 (25).
N-(4-Butylphenyl)-4-oxo-4H-chromene-2-carboxamide
(
16): 118.2 (C(8)), 121.1 (C(4’)), 124.4 (C(4a)), 126.2 (C(6)), 126.3
1
Yield: 69%. H NMR (CDCl3): δ = 0.93 (3H, t, J = 7.3, CH3), 1.31- (C(5)), 134.9 (C(7)), 139.0 (C(5’)), 148.1 (C(3’)), 150.1 (C(1’)),
1.41 (2H, m, CH2), 1.59-1.62 (2H, m, CH2), 2.61 (2H, t, J = 7.7, 154.0 (C(8a)), 155.2 (C(2)), 157.3 (CONH), 177.9 (C(4)). MS/EI
+
•
CH2). 7.21 (2H, d, J = 8.5, H(3’), H(5’)), 7.26 (1H, s, H(3)), 7.48
(1H, ddd, J = 1.0, 7.2, 8.1, H(6)), 7.57-7.63 (3H, m, H(8), H(2’),
H(6’)), 7.77 (1H, ddd, J = 1.6, 7.2, 8.5, H(7)), 8.24 (1H, dd, J =
1.6, 8.1, H(5)), 8.55 (1H, s, NH). 13C NMR (CDCl3): δ = 13.9
(CH3), 22.3 (CH2), 33.5 (CH2) 35.1 (CH2), 112.5 (C(3)), 118.1
(C(8)), 120.5 (C(2’), C(6’)), 124.4 (C(4a)), 126.1 (C(6)), 126.2
(C(5)), 129.2 (C(3’), C(5’)), 133.9 (C(4’)), 140.6 (C(1’)), 134.4
(C(7)), 154.7 (C(8a)), 155.2 (C(2)), 156.8 (CONH), 178.1 (C(4)).
m/z: 267 (22), 266 (M , 99), 238 (58), 237 (66), 210 (83), 89
(100).
N-(Furan-2-ylmethyl)-4-oxo-4H-chromene-2-carboxamide (28):
Yield: 35%. 1H NMR (CDCl3): δ= 4.68 (2H, d, J = 5.7, CH2), 6.42-
6.32 (2H, m, H(4’), H(5’)), 7.19 (1H, s, H(3)), 7.23 (1H, bs, NH),
7.42 (1H, dd, J = 0.9, 1.7, H(3’)), 7.48-7.43 (1H, m, H(6)), 7.52
(1H, dd, J = 0.5, 8.5, H(8)), 7.73 (1H, ddd, J = 1.7, 7.2, 8.7, H(7)),
8.22 (1H, dd, J = 1.4, 8.0, H(5)). 13C NMR (CDCl3): δ = 36.9 (CH2),
108.6 (C(5’)), 110.8 (C(4’)), 112.5 (C(3)), 118.2 (C(8)), 124.4
+
•
MS/EI m/z: 321 (M , 81), 278 (100), 107 (68), 89 (88).
N-(2-Methylphenyl)-4-oxo-4H-chromene-2-carboxamide
(17): (C(4a)), 126.1 (C(6)), 126.2 (C(5)), 134.7 (C(7)), 142.8 (C(3’)),
the synthetic procedure and structural elucidation data was 150.1 (C(1’)), 154.6 (C(8a)), 156.3 (C(2)), 159.2 (CONH), 178.2
described elsewhere.39
+
(C(4)). MS/EI m/z: 270 (22), 269 (M , 100), 297 (11), 146 (21),
•
N-(3-Methylphenyl)-4-oxo-4H-chromene-2-carboxamide
(18):
145 (16).
the synthetic procedure and structural elucidation data was
described elsewhere.39
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N-(Thiazol-2-yl)-4-oxo-4H-chromene-2-carboxamide
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DOI: 10.1039/C5RA14988F
ARTICLE
(
29): NMR (CDCl3): δ = 18.0 (CH3), 55.4 (OCH3), 111.8 (C(3)), 112.4
1
Yield: 55%. H NMR (CDCl3): δ = the synthetic procedure and (C(6’)), 116.2 (C(5’)), 118.0 (C(8)), 124.3 (C(4a)), 125.0 (C(6)),
structural elucidation data was described elsewhere.41
126.1 (C(3’)), 126.1 (C(5)), 127.0 (C(1’)), 132.2 (C(2’)), 134.6
(C(7)), 154.8 (C(8ª)), 155.1 (C(2)), 157.1 (C(4’)), 157.8 (CONH),
N-(5-Methylthiazol-2-yl)-4-oxo-4H-chromene-2-carboxamide
+
178.0 (C(4)). MS/EI m/z: 310 (23), 309 (M , 100), 195 (26), 136
•
(
30): the synthetic procedure and structural elucidation data
was described elsewhere.41
(72).
N-(4,5-dimethylthiazol-2-yl)-4-oxo-4H-chromene-2-
carboxamide (31): the synthetic procedure and structural
elucidation data was described elsewhere.41
Ethyl
4-methyl-2-(4-oxo-4H-chromene-2-
32): the synthetic
carboxamido)thiazole-5-carboxylate
(
procedure and structural elucidation data was described
elsewhere.41
Ethyl
4-methyl-2-(4-oxo-4H-chromene-3-
carboxamido)thiazole-5-carboxylate (35): yield: 15%. 1H NMR
(CDCl3): δ = 1.37 (3H, t, J = 7.1, OCH2CH3), 2.69 (3H, s, CH3),
4.33 (2H, q, J = 7.1, OCH2CH3), 7.57 (1H, m, H(6)), 7.61 (1H, d, J
= 8.4, H(8)), 7.82 (1H, ddd, J = 1.6, 7.3, 8.6, H(7)), 8.37 (1H, dd, J
= 1.6, 8.0, H(5)), 9.07 (1H, s, H(2)), 12.65 (1H, s, NH). 13C NMR
(CDCl3): δ = 14.4 (OCH2CH3) , 17.3 (CH3), 60.8 (OCH2), 114.1
(C(3)), 116.6 (C(3’)), 118.5 (C(8)), 123.9 (C(4a)), 126.6 (C(6)),
126.9 (C(5)), 135.3 (C(7)), 156.1 (C(8a)), 157.1 (C(4’)), 158.4
(C(1’)), 161.0 (CONH), 162.8 (COOCH2), 163.3 (C(2)), 176.5
Method C: To a solution of chromone-3-carboxylic acid (500
mg, 2.6 mmol) in DMF (4 mL), POCl3 (241 ꢀl, 2.6 mmol) was
added. The mixture was stirred at room temperature for 30
min, with the formation in situ of the corresponding acyl
chloride. Then the heteroaromatic amine was added. After 1-5
hours, the mixture was diluted with dichloromethane (20 mL),
washed with H2O (2X10 mL) and with saturated NaHCO3
solution (2X10 mL). The organic phase was dried with Na2SO4,
filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography or
crystallization.
+
•
(C(4)). MS/EI m/z: 359 (M+1, 64), 358 (M , 98), 313 (37), 174
(73), 173 (93), 121 (100).
Method B: To a solution of chromone-2-carboxylic acid (1.1
mmol) in DMF (1.5 mL), POCl3 (1 mmol) was added. The
mixture was stirred at room temperature for 30 min, with the
formation in situ of the corresponding acyl chloride. Then the
aromatic amine or the heteroaromatic amine was added. The
system was heated at 120 °C for 5 min in a microwave
apparatus. Subsequently, the mixture was poured in a beaker
and water was added. The formed solid was filtered and
purified by recrystallization (CH2Cl2/n-Hexane) or flash
chromatography (AcOEt/n-hexane).
N-(5-Methylthiazol-2-yl)-4-oxo-4H-chromene-3-carboxamide
1
33): yield: 15%. H NMR (CDCl3): δ = 2.43 (3H, d, J = 1.2, CH3),
(
7.19 – 7.12 (1H, m, H(4’)), 7.54 (1H, ddd, J = 1.0, 7.2, 8.1, H(6)),
7.59 (1H, dd, J = 0.6, 8.5, H(8)), 7.80 (1H, ddd, J = 1.7, 7.2, 8.7,
H(7)), 8.35 (1H, dd, J = 1.5, 8.0, H(5)), 9.05 (1H, s, H(2)), 12.43
(1H, s, NH). 13C NMR (CDCl3): δ= 11.7 (CH3), 114.6 (C(3)), 118.6
(C(8)), 124.1 (C(4a)), 126.7 (C(6)), 126.9 (C(5)), 128.4 (C(3’)),
135.2 (C(7)), 135.4 (C(4’)), 155.5 (C(1’)), 156.2 (C(8a)), 160.3
+
•
(CONH), 163.0 (C(2)), 176.7 (C(4)). MS/EI m/z: 286 (M , 91),
174 (30), 173 (100), 121 (61).
N-(2-Methoxyphenyl)-4-oxo-4H-chromene-2-carboxamide
N-(4,5-Dimethylthiazol-2-yl)-4-oxo-4H-chromene-3-
1
carboxamide (34): yield: 15%. H NMR (CDCl3): δ = 2.28 (1H, s,
(
19): the synthetic procedure and structural elucidation data
was described elsewhere.28
CH3), 2.32 (1H, s, CH3), 7.54 (1H, ddd, J = 1.0, 7.2, 8.1, H(6)),
7.59 (1H, dd, J = 0.6, 8.5, H(8)), 7.79 (1H, ddd, J = 1.7, 7.2, 8.7,
H(7)), 8.35 (1H, dd, J = 1.6, 8.0, H(5)), 9.04 (1H, s, H(2)), 12.37
(1H, s, NH). 13C NMR (CDCl3): δ = 11.0 (CH3), 14.7 (CH3), 114.7
(C(3)), 118.6 (C(8)), 120.9 (C(3’)), 124.1 (C(4a)), 126.7 (C(6)),
126.8 (C(5)), 135.2 (C(5)), 143.0 (C(4’)), 152.9 (C(1’)), 156.2
(C(8a)), 160.3 (CONH), 163.0 (C(2)), 176.7 (C(4)). MS/EI m/z:
N-(3-Methoxyphenyl)-4-oxo-4H-chromene-2-carboxamide
(
20): the synthetic procedure and structural elucidation data
was described elsewhere.28
N-(2,3-Dimethylphenyl)-4-oxo-4H-chromene-2-carboxamide
23): Yield: 68%. 1H NMR (CDCl3): δ = 2.15 (3H, s, CH3), 2.32
(
(3H, s, CH3), 6.96 (1H, s, H(3)), 7.25-7.12 (3H, m, H(4’), H(5’),
H(6’)), 7.58 (1H, ddd, J = 1.08, 7.18, 8.07, H(6)), 7.83 (1H, dd, J
= 0.58, 8.41, H(8)), 7.94 (1H, ddd, J = 1.70, 7.11, 8.68, H(7)),
8.10 (1H, dd, J = 1.41, 7.99, H(5)), 10.60 (1H, s, NH). 13C NMR
(CDCl3): δ = 14.2 (CH3), 20.0 (CH3), 110.8 (C(3)), 118.9 (C(8)),
123.6 (C(4a)), 124.4 (C(6')), 124.9 (C(6)), 125.4 ( C(5’)), 126.0
(C(5)), 128.2 (C(4’)), 132.7 (C(2’)), 134.6 (C(1’)), 135.0 (C(7)),
137.0 (C(3’)), 155.1 (C(8a)), 155.7 (C(2)), 157.9 (CONH), 177.2
+
•
302 (M+2), 300 (M , 100), 199 (45), 139 (73).
Pharmacology
CHO Membrane Preparation. All methods involving
membrane preparation for radioligand binding experiments
followed the procedures as described earlier.32 Membranes for
radioligand binding were prepared from CHO cells stably
transfected with the human adenosine receptor subtypes A1,
A2A, and A3 in a two-step procedure. In the first low-speed step
(1,000g for 10 min at 4°C), the cell fragments and nuclei were
removed. After that, the crude membrane fraction was
sedimented from the supernatant at 100,000g for 30 min. The
membrane pellet was then resuspended in the specific buffer
used for the respective binding experiments, frozen in liquid
nitrogen, and stored at -80ºC. For the measurement of the
+
•
(C(4)). MS/EI m/z: 293 (M , 40), 292 (100), 264 (13), 120 (21).
N-(4-Methoxy-2-methylphenyl)-4-oxo-4H-chromene-2-
1
carboxamide (25): Yield: 64%. H NMR (CDCl3): δ = 2.35 (3H, s,
CH3), 3.79 (3H, s, OCH3), 6.81-6.75 (2H, m, H(3’), H(5’)), 7.22
(1H, s, H(3)), 7.47 (1H, appt, J = 8.0, 8.0, H(6)), 7.56 (1H, d, J =
8.1, H(8)), 7.72 (1H, d, J = 8.4, H(6’)), 7.76 (1H, ddd, J = 1.7, 7.2,
8.7, H(7)), 8.23 (1H, dd, J = 8.0, 1.6, H(5)), 8.43 (1H, s, NH). 13C
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ARTICLE
adenylyl cyclase activity in A2B receptor expressing CHO cells, both proteins was carried out considering highly conserved
only one step of centrifugation was used in which the residues of the transmembrane helices. The same alignment
homogenate was sedimented for 30 min at 54,000g. The described by Katritch et al. was followed.43 The quality of the
resulting crude membrane pellet was resuspended in 50 mM geometry in the protein homology models was assessed using
Tris/HCl, pH 7.4 and immediately used for the adenylyl cyclase the Protein Geometry module in MOE that takes into account
assay.
Phi-Psi dihedral plots, bond lengths, bond angles, dihedrals,
side chain rotamers and non-bonded contacts. The presence of
conserved disulfide bridges between adenosine receptors was
checked, such as the disulfide bridge in hA3 between Cys166
(EL2) and Cys83 (TM3). An optimization of the protein pocket
using the Induce Fit Docking workflow was carried out.36 This
procedure included docking of high affinity ligands through
Glide and protein active site optimization with Prime. Different
hA3 models were tested, in a similar way as Katritch et al.,43
according to their potential to discriminate ligands from
decoys and hA3 high affinity compounds from other adenosine
sub-types ligands. Areas under the ROC curves (AUROCs) were
calculated showing results for some models greater than 0.80.
The best hA3 models were selected to run molecular docking
calculations.
Human Cloned A1, A2A, A3 Adenosine Receptor Binding Assay.
All Binding of [3H]CCPA to CHO cells transfected with the
human recombinant A1 adenosine receptor was performed as
previously described.32 Competition experiments were
performed for 3h at 25°C in 200 μL of buffer containing 1 nM
[3H] CCPA, 0.2 U/mL adenosine deaminase, 20 μg of
membrane protein in 50 mM Tris/HCl, pH 7.4 and tested
compound in different concentrations. Nonspecific binding
was determined in the presence of 1 mM theophylline. Binding
of [3H]NECA to CHO cells transfected with the human
recombinant A2A adenosine receptors was performed
following the conditions as that described for the A1 receptor
binding.32 In the competition experiments, samples containing
a protein concentration of 50 μg, 10 nM [3H]NECA and tested
compound in different concentrations were incubated for 3h
at 25°C. Nonspecific binding was determined in the presence of
100 μM R-PIA (R-N6-phenylisopropyladenosine). Binding of
[3H]HEMADO to CHO cells transfected with the human
recombinant A3 adenosine receptors was carried out as
previously described.32 The competition experiments were
performed for 3h at 25 °C in buffer solution containing 1 nM
[3H]HEMADO, 20 μg membrane protein in 50mM Tris-HCl, 1
mM ethylenediaminotetraacetate (EDTA), 10 mM MgCl2, pH
8.25 and tested compound in different concentrations.
Nonspecific binding was determined in the presence of 100
μM R-PIA.
Molecular Docking of Adenosine Receptors Ligands (hA2A and hA3)
Molecular docking simulations were carried out using the
Schrödinger package.36 The database of chromone derivatives
was pre-processed with LigPrep module. The structures were
optimized and different tautomers and protonation states
(pH=7.0±2.0) were generated. Protein structures were also
prepared with the Protein Preparation Wizard workflow.36 This
process includes different steps, i.e., assigned bond orders,
added cap termini, optimized the hydrogen-bonding network
and protonation states of some residues, among others. The
molecular docking using the hA2A crystallized structure (PDB
code: 3EML)35 and the homology model for the hA3 was carried
out. No water molecules in the pocket of hA3 were included in
the docking calculations. For the hA2A, calculations without
water and with water were performed. The compounds were
docked with Glide module. Selection of the described binding
modes was carried out taking into account the energetic
parameter Emodel and number of similar poses yielded by the
calculations.
Adenylyl Cyclase Activity. Due to the lack of a suitable
radioligand for hA2B receptor in binding assay, the potency of
antagonists at A2B receptor (expressed on CHO cells) was
determined in adenylyl cyclase experiments instead. The
procedure was carried out as described previously with minor
32
modifications.31,
Membranes were incubated with about
150000 cpm of [α-32P]ATP for 20 min in the incubation mixture
as described, without EGTA and NaCl. For agonists, the EC50
values for the stimulation of adenylyl cyclase were calculated
with the Hill equation. Hill coefficients in all experiments were
near unity. IC50 values for concentration-dependent inhibition
of NECA-stimulated adenylyl cyclase caused by antagonists
were calculated accordingly. Dissociation constants (Ki) for
antagonist were then calculated from the Cheng and Prusoff
equation.33
Acknowledgements
The authors would like to thank Fundação para a Ciência e
Tecnologia (FCT) -Pest/C-QUI/UI0081/2013- and “Angeles
Alvariño, Plan Galego de Investigación, Innovación
e
Crecemento 2011-2015 (I2C)” for the financial support. Thanks
are due to Fundação para a Ciência e Tecnologia (FCT),
Programa Operacional Potencial Humano (POPH) and Quadro
de Referência Estratégica Nacional (QREN) for the post-
Molecular Modeling
A homology model of hA3 was constructed using MOE doctoral
and
doctoral
grants:
A.
Gaspar
software.43 Detailed description of the homology models are
provided in previous publications of our research group.21 The
crystal structure of the hA2A adenosine receptor (PDB: 3EML)35
was used as a template in the modeling. Alignment between
(SFRH/BPD/93331/2013), F. Cagide (SFRH/BPD/74491/2010)
and J. Reis (SFRH/BD/96033/2013).
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29 J. Reis, A. Gaspar, F. Borges, L. R. Gomes and J. N. Low, J.
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Fredholm and M. J. Lohse, Naunyn. Schmiedebergs Arch.
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