Structure-Affinity Relationships and Structure-Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione Derivatives as Human Adenosine A3 Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.7b00950

We expanded on a series of pyrido[2,1-f]purine-2,4-dione derivatives as human adenosine A₃ receptor (hA₃R) antagonists to determine their kinetic profiles and affinities. Many compounds showed high affinities and a diverse range of k

Full text of DOI:10.1021/acs.jmedchem.7b00950

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Article
Structure-Affinity Relationships and Structure-
Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione
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Derivatives as Human Adenosine A Receptor Antagonists
Lizi Xia, Wessel A. C. Burger, Jacobus P. D. van Veldhoven, Boaz J. Kuiper, Tirsa T. van
Duijl, Eelke B. Lenselink, Ellen Paasman, Laura H. Heitman, and Adriaan P. IJzerman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00950 • Publication Date (Web): 14 Aug 2017
Downloaded from http://pubs.acs.org on August 14, 2017
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StructureꢀAffinity Relationships and Structureꢀ
Kinetics Relationships of Pyrido[2,1ꢀf]purineꢀ2,4ꢀ
dione Derivatives as Human Adenosine A3 Receptor
Antagonists
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Lizi Xia, Wessel A. C. Burger, Jacobus P.D. van Veldhoven, Boaz J. Kuiper, Tirsa T. van Duijl,
Eelke B. Lenselink, Ellen Paasman, Laura H. Heitman, and Adriaan P. IJzerman*
Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden
University, 2300 RA Leiden, The Netherlands
ABSTRACT: We expanded on a series of pyrido[2,1ꢀf]purineꢀ2,4ꢀdione derivatives as human
adenosine A3 receptor (hA3R) antagonists to determine their kinetic profiles and affinities. Many
compounds showed high affinities and a diverse range of kinetic profiles. We found hA3R
antagonists with very short residence time (RT) at the receptor (2.2 min for 5) and much longer
RTs (e.g. 376 min for 27, or 391 min for 31). Two representative antagonists (5 and 27) were
tested in [35S]GTPγS binding assays, and their RTs appeared correlated to their (in)surmountable
antagonism. From a konꢀkoffꢀKD kinetic map we divided the antagonists into three subgroups,
providing a possible direction for the further development of hA3R antagonists. Additionally, we
performed a computational modelling study that sheds light on the crucial receptor interactions
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dictating the compounds’ binding kinetics. Knowledge of target binding kinetics appears useful
for developing and triaging new hA3R antagonists in the early phase of drug discovery.
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INTRODUCTION
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The adenosine A3 receptor is the youngest member discovered in the family of adenosine
receptors,1 all of which belong to class A Gꢀprotein coupled receptors (GPCRs) and fall into four
distinct subtypes (A1, A2A, A2B and A3). Although all subtypes are activated by the endogenous
ligand adenosine, these purinergic receptors differ from each other in their distribution and to
which G protein they are coupled. Following agonist activation, the A1 and A3 adenosine
receptors cause a decrease in cAMP levels as they primarily couple to Gi proteins. The A2A and
A2B adenosine receptors on the other hand, are primarily linked to Gs proteins and this leads to
increased levels of cAMP upon receptor activation.2
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Although the pharmacological characterization of adenosine receptors has been well
documented,3 the human adenosine A3 receptor (hA3R) is less well characterized because of its
“dichotomy” in different therapeutic applications.4 Moreover, certain ligands have been
described as cytoprotective or cytotoxic merely depending on the concentration employed,
highlighting the difficulties that arise when characterizing novel hA3R compounds.5
Nevertheless, there is no doubt that the hA3R has therapeutic potential in clinical indications (i.e.
cardiovascular diseases,6, 7 cancer,7, 8 and respiratory diseases7, 9ꢀ11), due to its overexpression on
cancer and inflammatory cells.3, 12ꢀ15
Traditional drug screening methods, and those employed in previous hA3R drug discovery
attempts, revolve around the use of a ligand’s affinity as the selection criterion for further
optimization in a soꢀcalled structureꢀaffinity relationships (SAFIRs) approach. In recent years,
however, there has been emerging the realization that selecting ligands based on their affinity, an
equilibrium parameter, does not necessarily predict in vivo efficacy. This is due to the dynamic
conditions in vivo, that often are in contrast to the equilibrium conditions applied in in vitro
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assays.16 In fact a ligand’s kinetic properties may provide a better indication of how a ligand will
perform in vivo.17 Specifically, the parameter of residence time (RT) has been proposed as a
more relevant selecting criterion. The RT reflects the lifetime of the ligandꢀreceptor complex and
can be calculated as the reciprocal of the ligand’s dissociation constant (RT=1/koff).18, 19
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While the binding kinetics of some (labeled) hA3R agonists have been studied,20 this
parameter has not been part of medicinal chemistry efforts for antagonists, i.e. yielding structureꢀ
kinetics relationships (SKRs), next to SAFIRs.21 Therefore, in order to provide the first SKR
analysis on the hA3R, a highly potent and selective hA3R antagonist scaffold was chosen. The
pyrido[2,1ꢀf]purineꢀ2,4ꢀdione template has been previously characterized with respect to affinity
alone. In a Topliss approach22 we had synthesized and characterized a number of highly potent
and selective hA3R antagonists.23, 24 One of the reference antagonists (1) with good affinity and
selectivity over other adenosine receptors is represented in Table 1. Using this compound as the
starting point, we further selected and synthesized compounds to add to the library of pyrido[2,1ꢀ
f]purineꢀ2,4ꢀdione derivatives. Using radioligand displacement assays and competition
association assays, we obtained affinity (Ki) and kinetic parameters (kon, koff, and RTs). This
allowed a full SKR study alongside a more traditional SAFIR analysis. The findings provide
information on the structural requirements for a favorable kinetic profile at the hA3R and
consequently may improve the in vitro to in vivo translation for hA3R antagonists.
RESULTS AND DISCUSSION
Chemistry. The synthesis approach shown in Scheme 1 was adapted from Priego et al.23, 24
Starting from the commercially available materials benzylurea (3), ethyl cyanoacetate and
sodium methoxide 1ꢀbenzylꢀ6ꢀaminoꢀuracil (4) was synthesized in a 88% yield.25 In situ
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dibromination of uracil 4 at the C5 position by Nꢀbromosuccinimide, followed by cyclisation
with 4ꢀmethoxypyridine gave the pyrido[2,1ꢀf]purineꢀ2,4ꢀdione (5) in a one pot reaction. Final
compounds 1, 2 and 6-22 (as depicted in Table 1) were obtained, with yields varying in the range
of 3ꢀ86%, by alkylating the N3 position of 5 using a variety of alkyl, alkenyl and alkynyl
bromides in acetonitrile and 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU) as a base. Secondly, to
be able to diversify on the N1 (R2) position, building block 23 had to be obtained. Full
conversion of methylcyclopropyl compound 2 into the desired debenzylated 23 was realized by
multiple additions of ammonium formate and Pd(OH)2 at 80 oC in ethanol overnight. Due to poor
solubility 23 was extracted with hot DMF and Pd(OH)2 was removed by filtration, resulting in a
quantitative yield. Finally various N1 substituted benzyl (24-32) and phenethyl (33) derivatives
(Scheme 1) were made starting from the respective benzylꢀ or phenethyl bromides in DMF with
K2CO3 used as base.
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Biological Evaluation. All binding affinities of the pyrido[2,1ꢀf]purineꢀ2,4ꢀdione derivatives
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were determined at 25 C in a 2 hours incubation protocol. All compounds were able to
concentrationꢀdependently
inhibit
specific
[3H]8ꢀEthylꢀ4ꢀmethylꢀ2ꢀphenylꢀ(8R)ꢀ4,5,7,8ꢀ
tetrahydroꢀ1Hꢀ imidazo[2,1ꢀi]ꢀpurinꢀ5ꢀone26 ([3H]PSBꢀ11, 34) binding to the human adenosine
A3 receptor and their affinities are listed in Tables 1, 2 and 3. All compounds had
(sub)nanomolar binding affinities ranging from 0.38 nM for compound 27 to 108 nM for
compound 5.
Subsequently, the human adenosine A3 receptor ligands were screened in a soꢀcalled “dualꢀ
point” competition association assay,27 allowing for the semiꢀquantitative estimation of the
compounds’ dissociation rates and therefore the compounds’ RTs. The specific binding of
[3H]34 was measured after 20 and 240 minutes in the absence and presence of a single
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concentration (i.e. 1 x IC50) of unlabeled human adenosine A3 receptor antagonists, which
yielded their Kinetic Rate Index (KRI). A long RT compound shows a characteristic “overshoot”
followed by a steady decrease in specific binding until a new equilibrium is reached; in such a
case the KRI value is greater than unity. Conversely, a ligand with a fast dissociation rate is
represented by a more shallow curve, yielding a KRI value smaller than one when dividing the
binding at t1 by the binding at t2. The KRI values in the series ranged from 0.38 to 4.06 (Table 1,
2 and 3).
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Compounds with a KRI value less than 0.7 or greater than 1.5 were selected for complete
kinetic characterization through the use of a competition association assay with [3H]34 (Figure
1A). In order to obtain extensive StructureꢀKinetics Relationships (SKRs), close structural
analogs ( 9, 28, 29 and 30) of 1 were also tested to obtain their association (kon) and dissociation
(koff) rate constants. Association rate constants varied by 30ꢀfold, ranging from (1.0 ± 0.1) x 105
Mꢀ1sꢀ1 for antagonist 31 to (3.0 ± 0.3) x 106 Mꢀ1sꢀ1 for antagonist 30 (Table 3). Interestingly, there
was an approximately 290ꢀfold difference in dissociation rate constants, reflecting the divergent
KRI values. Antagonist 5 had the fastest dissociation rate constant of (1.4 ± 0.5) x 10ꢀ2 sꢀ1 and
thus the shortest RT of 2.2 min, while both antagonist 27 and 31 had the slowest dissociation rate
constants of (4.7 ± 0.7) x 10ꢀ5 sꢀ1 and (5.3 ± 1.5) x 10ꢀ5 sꢀ1, respectively, and thus the longest RTs
of 376 min and 391 min, respectively. Of note, the long RT antagonist 27 (Figure 1A) displayed
a typical “overshoot” in the competition association curve, indicative of a slower dissociation
than the radiolabeled probe [3H]34, while the short RT antagonists, exemplified by antagonist 5
(Figure 1A) presented more shallow, gradually ascending curves. There was a good correlation
between the negative logarithm of the antagonists’ dissociation rate constants and their KRI
values derived from the kinetic screen (Figure 2A), which confirmed that a compound’s KRI
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value is a good predictor for its dissociation rate constant. Notably, the experimental
temperatures in the kinetic assays were lower than in the equilibrium displacement assays (25 oC
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compounds tested. This is shown in Figure 1B, where the “overshoot” of long RT antagonist 27
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happened before the t1 checkpoint of 20 min, which did not happen at 10 C. A significant
correlation was also observed between the antagonist affinities (Ki values) determined in
equilibrium displacement experiments and their kinetic KD values derived from competition
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association experiments (Figure 2B), despite the differences in assay temperature (25 C vs 10
oC). Interestingly, the kinetic association rate constants (kon) did not show any significant
correlation with affinity (Figure 2C), whilst the dissociation rate constants (koff) had a fair
correlation with affinity (Figure 2D).
The representative long RT and short RT antagonists (27 and 5) were selective for the hA3
receptor when compared to other adenosine receptors (i.e. human adenosine A1 and A2A receptor,
Table S1). These two antagonists (27 and 5) with comparable association rate constants but
distinct dissociation rate constants (or RTs) were further analyzed in a [35S]GTPγS binding assay
in which we studied the (in)surmountable antagonism induced by the two compounds (Figure 3).
Moreover, a konꢀkoffꢀKD “kinetic map” (Figure 4) was constructed based on the compounds’
divergent affinities (expressed as kinetic KD values) and kinetics parameters, yielding a division
of these antagonists into three different subꢀcategories: antagonists that show similar koff values
(< 2ꢀfold) but due to differing kon values (> 28ꢀfold) have different KD values (~100ꢀfold, Group
A); antagonists that display similar KD values (< 10ꢀfold) despite showing divergent koff and kon
values (17ꢀfold and 30ꢀfold, Group B); antagonists with similar kon values (< 5ꢀfold), but due to
differing koff values (~290ꢀfold) have different KD values (> 110ꢀfold, Group C). Additionally,
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we applied molecular modeling to compare the binding behavior in some molecular detail of
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several antagonists with similar affinities (2 vs 10; 31 vs 29 or 30) (Figure 5).
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Structure-Affinity Relationships (SAFIRs) and Structure-Kinetics relationships (SKRs).
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According to previous studies from our group,23, 24 methoxyꢀsubstitution at the 8 position (Table
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1) of the pyrido[2,1ꢀf]purineꢀ2,4ꢀdione scaffold yielded selective hA3R antagonists with good
affinity (3.2 nM for 1 as a reference compound). From our preliminary studies, this methoxyꢀ
group appeared important for slow dissociation (1 vs Compound S2 from Figure S1, Table S2).
Due to the nanomolar affinity and closeꢀtoꢀunity KRI value of 1, it was treated as the starting
point of this SAFIR and SKR study, having, on further analysis, an association rate constant of
(8.5 ± 1.2) x 105 Mꢀ1sꢀ1 and a dissociation rate constant of (3.2 ± 0.02) x 10ꢀ4 sꢀ1 (RT = 52 min).
Next, we decided to investigate R1 substitutions (Table 2), beginning with antagonist 5 (R1 = H)
The substitutions at R1 (Table 2). Firstly, an increase in alkyl chain length was investigated,
indicating an elongated carbon chain had a cumulative effect on KRI (5, 6, 7, 8, 10, 11), with the
exception of antagonist 9 (KRI values from 0.38 to 4.06). One could point to a possible
correlation between lipophilicities and dissociation rate constants (and consequently RTs) to
explain this trend (Figure S2A). However, with all of the antagonists kinetically characterized,
no such correlation was observed (Figure S2B). Therefore other reasons should be taken into
account as to why elongating the carbon chain has such a profound effect on the ligand’s
dissociation rate. The role of membraneꢀdrug interactions in determining the pharmacological
profile is a possible reason, especially the role long carbon tails have in such interactions.28
Interesting to point out is the affinity of antagonist 11 which, had a traditional lead selection
process taken place, would most likely have resulted in the elimination of this compound due the
more favorable affinity and hydrophilic properties of its shorter carbon chain counterparts (8 or
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9) (affinities: 6.8 nM vs 1.5 nM or 3.5 nM, KRI values: 4.06 vs 1.29 or 1.11). This would have
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overlooked the efficacy this compound could offer due to its longer residence time.
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Secondly, the presence of a more rigid substitution of the R1 group of saturated equivalents
(antagonists 1 and 8) led to antagonists 12 and 14, with similar improvement in the affinity pairs
(12 and 14: 5.9 nM and 1.4 nM; 1 and 8: 3.2 nM and 1.5 nM) and KRI values (12 and 14: 0.72
and 1.23; 1 and 8: 0.99 and 1.29). Further rigidification with alkyne (13) rather than alkene (12)
maintained affinities (4.3 nM vs 5.9 nM) and increased KRI values (1.20 vs 0.72). This alkyne
could be the starting point for a further study on “clickꢀchemistry” for introducing e.g.,
fluorescent tags.29ꢀ31
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Thirdly, the introduction of a polar atom or group in antagonist 15 or 16, respectively, led to a
decrease in affinity compared to their nonꢀpolar counterpart 1 (23 nM or 81 nM vs 3.2 nM). The
changes in KRI values between antagonist 1 and its polar counterparts 15 and 16 can be
considered minor (0.99 vs 0.70 and 1.04). Of note, by comparing affinities and kinetic profiles of
polar antagonist 15 with its nonꢀpolar equivalent 1, we found the polarity at the “lipophilic
carbon chain” resulted in slower association (kon of (4.3 ± 0.8) x 105 Mꢀ1sꢀ1 vs (8.5 ± 1.2) x 105
Mꢀ1sꢀ1) but faster dissociation (koff of (6.3 ± 0.7) x 10ꢀ4 sꢀ1 vs (3.2 ± 0.02) x 10ꢀ4 sꢀ1), with a
concomitant decrease in affinity (23 nM vs 3.2 nM).
Moreover, the bulkiness of the substituents was studied with branched carbon side chains (17,
18, 19, 20 and 21) or aliphatic rings (2 and 22). As to the branched carbon side chains,
compound affinities remained in the nanomolar range, while in terms of KRI values, 2ꢀcarbonꢀ
linker branched side chains (17 and 18) caused larger KRI values than those of either their linear
counterparts (8 and 9) or 3ꢀcarbonꢀlinear branched side chains (19 and 20) (17: 1.64 vs 1.29 or
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1.39; 18: 1.73 vs 1.11 or 0.95). Although the association rate constants of 17 and 18 were
similar to other antagonists in Table 2, the dissociation rate constants suggest their branched side
chains have an extra “anchoring” effect, compared with the linear counterparts. For example, the
koff of 18 with a 5ꢀcarbon branched side chain was quite similar to 10 or 11 having a 6 or 7ꢀ
carbon linear side chain ((1.1 ± 0.4) x 10ꢀ4 sꢀ1 vs (8.2 ± 1.3) x 10ꢀ5 sꢀ1 or (6.2 ± 0.2) x 10ꢀ5 sꢀ1).
The presence of a slightly less polar but larger silicon atom (21) instead of carbon (18) made the
KRI value decrease (1.36 vs 1.73), although the affinity remained virtually the same (2.7 nM vs
3.5 nM).
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Interestingly, another reported analogue (2)23 of compound 1, with cyclopropylmethyl
substitution at the R1 group, led to unique kinetic parameters, i.e. a combination of a fast
association rate constant (((2.8 ± 0.5) x 106 Mꢀ1sꢀ1 vs (8.5 ± 1.2) x 105 Mꢀ1sꢀ1) and a slow
dissociation rate constant ((6.0 ± 1.7) x 10ꢀ5 sꢀ1 vs (3.2 ± 0.02) x 10ꢀ4 sꢀ1), although the affinities
of 2 and 1 were similar (1.0 ± 0.03 nM vs 3.2 ± 0.1 nM, respectively). The RT of compound 2
was the longest in Table 2 with 315 min. For the antagonist with cyclobutylmethyl (22), affinity
(2.7 nM vs 1.0 nM) and KRI value (1.48 vs 2.68) were lower than for compound 2.
Although the dissociation rate constants of the antagonists in Table 2 varied greatly depending
on the R1 substituent, the association rate constants were more similar (within 5ꢀfold).
Association rate constants are often reasoned to be caused by a diffusion limited process
whereby the collision rate of ligand and receptor determines the rate of ligandꢀreceptor complex
formation.32 When no conformational changes are required for the receptor and ligand to bind
and when taking into account the proportion of the receptor responsible for binding, this sets the
association rate constant at observed limits of around 107 Mꢀ1sꢀ1.33 As the association rate
constants for all R1 substituted compounds were slower than the diffusion limit by at least 3.5
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fold (2), we hypothesize target engagement for R1 substituted antagonists is more hampered than
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The substitutions at R2ꢀgroup (Table 3). From Table 2 we learned that cyclopropylmethylꢀ
substituted antagonist 2 exhibited a kinetic profile as a long RT compound whilst showing the
affinity previously reported.23 As a result, this compound became the starting point for our
exploration of the substitutions (R2ꢀgroup) on the aromatic ring.
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Introduction of a nonꢀpolar alkyl substituent on antagonist 2’s benzyl ring (24, 25, 26), resulted
in a decrease in KRI values (from 2.68 to 0.81), while slight variations in affinity were observed.
Then, introduction of a polar methoxy substituent on antagonist 2’s benzyl ring, led to mixed
results with a small decrease in RT at paraꢀposition, and a slight increase in RT at metaꢀposition
in 28 (250 min vs 315 min) and 27 (376 min vs 315 min), respectively. In particular, the long
residence time for 27 in combination with its subꢀnanomolar affinity (0.38 nM), made this
compound stand out in the series.
Next, halogen substitutions on antagonist 2’s benzyl ring were examined. Apparently, the
position of halogen substitution is important for affinity as paraꢀsubstitution in antagonist 30 and
32 yielded similar affinity compared to 2 (1.2 nM vs 1.2 nM vs 1.0 nM). The one compound with
metaꢀsubstitution, 29, showed a 5ꢀfold decrease in affinity compared to 2 (4.9 nM vs 1.0 nM).
Dichloroꢀsubstituted compound 31 had the largest KRI value (3.12) among the halogenꢀ
substituted antagonists; the paraꢀbromo substituted compound 32 was similar in this respect to
paraꢀchloro substituted 30 (1.19 vs 1.11). In a full competition association experiment we
determined the rate constants for 31, and learned it had the longest RT of all compounds
kinetically characterized (391 min), concomitant with the slowest association rate constant of the
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compounds kinetically characterized ((1.0 ± 0.1) x 105 Mꢀ1sꢀ1). Previous theoretical studies have
indicated the strength of halogen bonding can be increased through the introduction of electron
withdrawing groups onto halobenzenes.34 Such would be the case for 31 where the additional
chloro substituent forms a stronger halogen bonding interaction with the R2 binding pocket.
Introducing a phenethyl (33) rather than benzyl substituent (2) led to a decrease in affinity (7.7
nM vs 1.0 nM), whilst the KRI value was also strongly affected (1.09 vs 2.68). This observation
parallels our previous findings that the binding pocket for the R2 substituent is of limited size.23
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Functional Assay. Following kinetic characterization, a long (27) and a short (5) RT
compound were chosen for functional characterization in a [35S]GTPγS binding assay, also
because for these two compounds the kon values were similar (4.8 ± 0.2) x 105 Mꢀ1sꢀ1 vs (5.3 ±
1.5) x 105 Mꢀ1sꢀ1). This difference allowed a possible link to be made between RTs and
efficacies. Pretreatment of hA3 receptor membranes with increasing concentrations of the long
RT antagonist 27, before stimulation by the A3 receptor agonist 2ꢀClꢀIBꢀMECA, induced
insurmountable antagonism. In other words, the 2ꢀClꢀIBꢀMECA concentrationꢀeffect curves
were shifted to the right with a concomitant decrease in the maximal response (Figure 3A).
Conversely, the short RT antagonist 5 displayed surmountable antagonism, shifting 2ꢀClꢀIBꢀ
MECA’s curves to the right without affecting its maximum effect (Figure 3B). In this
experimental setꢀup, the Schildꢀslope of 5 generated from Schildꢀplots was close to unity (Table
4), and the compound’s pA2 value was comparable with its pKi value (6.8 ± 0.4 vs 7.0 ± 0.02).
We also performed coꢀincubation experiments with these antagonists in the presence of 2ꢀClꢀIBꢀ
MECA. In this experimental setꢀup, all antagonists produced a rightward shift of the 2ꢀClꢀIBꢀ
MECA concentrationꢀeffect curves without a suppression of the maximal response (Figure 3C
and D). Notably, the Schildꢀslopes of both long and short RT antagonists (27 and 5) were close
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to unity (0.9 ± 0.2 for 27, 1.0 ± 0.2 for 5, Table 4). In addition, the pA2 value of 5 was
comparable with the result from the preꢀincubation condition (7.2 ± 0.4 vs 6.8 ± 0.4, Table 4),
and the pA2 value of 27 was also in agreement with its pKi value (8.9 ± 0.3 vs 9.4 ± 0.03).
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Kinetic Map. Using the association (kon) and dissociation (koff) rate constants obtained from
competition association experiments (Tables 1ꢀ3), a kinetic map (Figure 4) was constructed by
plotting these values on the yꢀaxis and xꢀaxis, respectively. The dashed diagonal parallel lines
represent the kinetically derived KD values (KD = koff/kon). Out of this map three subgroups
emerged. Group A represents compounds that exhibit similar koff values, but with vastly different
kon values. As a consequence a diverse range of KD values was observed. Previous SKR studies
have primarily focused on optimizing dissociation rates and RTs for predicting inꢀvivo efficacy
and creating a kinetically favorable ligand. Yet recently, there has been greater acknowledgment
of the important role that the association rate constants may play in determining the efficacy of a
drug as the result of increased rebinding or increased drugꢀtarget selectivity.19 A kinetic map
would thus allow for the selection of compounds with appropriate RTs whilst exploring the role
of association rate constants in determining efficacy by choosing a rapidly or slowly associating
compound, i.e. 2 or 31 ((2.8 ± 0.5) x 106 Mꢀ1sꢀ1 vs (1.0 ± 0.1) x 105 Mꢀ1sꢀ1). Group B displays
ligands that exhibit a narrow range of affinity (KD: 0.1 nM ꢀ 1 nM), yet a wide range of koff
values that result in RTs ranging from 20 min to 391 min. This information would have gone
unnoticed in a traditional SAFIR hitꢀtoꢀlead approach and would most likely have led to the
selection of high affinity compounds not in possession of a potentially efficacy promoting long
residence time. Thus, combining SAFIR with SKR aspects in lead optimization would allow the
selection of not only potent but also long RT compounds through the drug development pipeline.
Lastly group C represents compounds that present similar kon values but due to differing koff
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values show considerable differences in affinities (KD). This illustrates the differences that were
observed in the binding kinetics of the R1 and R2 substituents, as group C mainly consists of R1
substituents (nonꢀcyclopropylmethyl substituents), whilst group A mainly consists of R2
substituents (cyclopropylmethyl substituents). This difference also suggests a different mode of
receptorꢀligand interaction during the binding process of the two ligand groups.
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Altogether, the construction of a kinetic map allows for a more detailed categorization of
compounds’ affinities as dictated by their kinetic rate constants. In previous studies, such a
separation has explained the different therapeutic effects molecules exhibit highlighting the
benefits of such an inꢀdepth analysis.35, 36
Given the putative link between RT and clinical efficacy, it may be postulated that the lack of
hA3R antagonists progressing from preclinical trials is due to insufficient selection criteria
employed in these initial phases of hA3R drug screening. As previously reported, hA3R
antagonists are reasoned to be beneficial in the treatment of Chronic Obstructive Pulmonary
Disease (COPD).37 For this indication, a number of antagonists are available that act at the
muscarinic M3 receptor.38 For these therapeutics their dosing regime and thus duration of action
have been linked to their RT. For example, aclidinium which requires a twice daily dosing
regimen, exhibits a much shorter RT than tiotropium that in turn requires only once daily
dosing.16 This extended duration of action that enables longꢀlasting efficacy and practical dosing
regimens at the muscarinic M3 receptor is thought to be a beneficial feature in the treatment of
chronic illnesses.39, 40 As hA3R antagonists can be used to treat chronic COPD but also a number
of other chronic disorders, we could imagine that considering the ligand’s kinetic profile early in
the drug screening process, would reduce the likelihood of failure due to insufficient efficacy in
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future clinical trials. Perhaps when selecting hA3R antagonists with a favorable long RT, i.e. the
group A in the kinetic map, will we see the therapeutic potential of the hA3R fulfilled.
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Computational studies. Finally, we decided to further investigate the ligandꢀreceptor
interactions using a homology model of the adenosine A3 receptor, based on the crystal structure
of the adenosine A2A receptor (PDB: 4EIY)41. WaterMap calculations were applied to try and
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explain the variance in kinetic profiles of different ligands by unfavorable hydration42, 43
.
Antagonist 2 (in black stick representation) was docked in the homology model. As a first step
it was placed inside the transmembrane bundle, with the tricyclic ring system surrounded by
TM3, TM6 and EL2. Hydrogen bonding was constrained between the amideꢀhydrogen (ꢀNH2,
δ+) from Asn2506.55 and the carbonylꢀoxygen (ꢀC=O, δꢀ) at the
C
4ꢀposition of the pyrido[2,1ꢀ
f]purineꢀ2,4ꢀdione scaffold (Figure 5, left). In order to compare differences between the ligands,
an “apo” WaterMap of the hA3 receptor was generated. Hydration sites shown as red and orange
spheres represent positions where “unstable” water molecules are found. Antagonist 10 (hexylꢀ
substitution), with comparable koff ((8.2 ± 1.3) x 10ꢀ5 sꢀ1 vs (6.0 ± 1.7) x 10ꢀ5 sꢀ1) to 2 but 10ꢀfold
slower kon ((2.3 ± 1.0) x 105 Mꢀ1sꢀ1 vs (2.8 ± 0.5) x 106 Mꢀ1sꢀ1), was docked with two different
binding modes in the same binding site (Figure 5 upꢀright, cyan and grey sticks). We found
additional unstable waters (8, 11, 22 in Figure 5 upꢀright) surrounding the lipophilic substituents
of the compounds, which could be explained as hindrance when the antagonist is associating
with the binding site.
The same WaterMap was used to investigate the kinetic profile of antagonist 31. Indeed,
hydration sites 6, 39, 42 and 45 are proposed to be displaced by the 3,4ꢀdichloro substituent.
Thus, both the association and dissociation of 31 were slowed down by these unstable waters.
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For the association process, the lipophilic 3,4ꢀdichloro moiety has difficulty in approaching the
occupied unstable hydration sites ((1.0 ± 0.1) x 105 Mꢀ1sꢀ1, slowest kon in the whole series); the
same lipophilic 3,4ꢀdichloro substituent seems to provide more stabilization to the receptorꢀ
ligand complex, thus hampering the dissociation process ((5.3 ± 1.5) x 10ꢀ5 sꢀ1, slowest koff in the
whole series). Interestingly, by removing a single chloro atom at either the 3ꢀ or 4ꢀ position on
the benzylꢀring (30 or 29), association and dissociation rate constants became faster by
approximately 10ꢀfold. Although the differences in their kon and koff values were modest (2ꢀ3
fold), the unstable hydration sites may prevent the 4ꢀchloroꢀsubstituted antagonist 30 from
reaching the hydration sites 6, 39 and 42 that interact with the 3ꢀCl substituent; consequently,
both its association and dissociation rate constants were faster than of the 3ꢀchloroꢀsubstituted
counterpart 29 (kon: (3.0 ± 0.3) x 106 Mꢀ1sꢀ1 vs (8.2 ± 1.3) x 105 Mꢀ1sꢀ1; koff: (8.2 ± 0.2) x 10ꢀ4 sꢀ1
vs (4.7 ± 0.7) x 10ꢀ4 sꢀ1).
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CONCLUSIONS
We have demonstrated that, next to affinity, additional knowledge of target binding kinetics is
useful for selecting and developing new hA3R antagonists in the early phase of drug discovery.
By introducing proper substituents at the N3 position or the N1 benzyl ring of a series of
pyridopurinediones, divergences in kinetic profiles were observed, while almost all compounds
had high and often similar affinity. Two representative ligands (5 and 27) were tested in
[35S]GTPγS binding assays, confirming the link between their RTs and their (in)surmountable
antagonism. According to these findings, a konꢀkoffꢀKD kinetic map was constructed and
subsequently the antagonists were divided into three subꢀgroups. Additionally, we also
performed a computational modelling study that sheds light on the crucial interactions (including
with water molecules) for both the association and dissociation kinetics of this family of
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antagonists. It should be mentioned that the kinetic parameters were derived at the hA3R, which
may be different in e.g., rodents used in advanced animal models. Still, this study suggests that
favorable long RTs would be a proper indicator in the development of hA3R antagonists for
chronic inflammatory conditions, e.g. COPD.
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EXPERIMENTAL SECTION
Chemistry. All solvents and reagents were purchased from commercial sources and were of
analytical grade. Distilled water will be referred to as H2O. TLC analysis was performed to
monitor the reactions, using Merck silica gel F254 plates. Grace Davison Davisil silica column
material (LC60A, 30–200 µm) was used to perform column chromatography. Microwave
reactions were performed in an Emrys Optimizer (Biotage AB, formerly Personal Chemistry). 1H
and 13C NMR spectra were recorded on a Bruker DMXꢀ400 (400 MHz) spectrometer, using
tetramethylsilane as internal standard. Chemical shifts are reported in δ (ppm) and the following
abbreviations are used: s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet. The
analytical purity of the final compounds is 95% or higher and was determined by highꢀ
performance liquid chromatography (HPLC) with a Phenomenex Gemini 3 µm C18 110A
column (50 4.6 mm, 3 µm), measuring UV absorbance at 254 nm. The sample preparation and
HPLC method was as follows: 0.3–0.6 mg of compound was dissolved in 1 mL of a 1:1:1
mixture of CH3CN/H2O/tꢀBuOH and eluted from the column within 15 min at a flow rate of 1.3
mL/min. The elution method was set up as follows: 1–4 min isocratic system of H2O/CH3CN/1%
TFA in H2O, 80:10:10; from the 4th min a gradient was applied from 80:10:10 to 0:90:10 within
9 min, followed by 1 min of equilibration at 0:90:10 and 1 min at 80:10:10. Liquid
chromatography–mass spectrometry (LC–MS) analyses were performed using a Thermo
Finnigan Surveyor—LCQ Advantage Max LC–MS system and a Gemini C18 Phenomenex
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column (50 4.6 mm, 3 µm). The elution method was set up as follows: 1–4 min isocratic system
of H2O/CH3CN/1% TFA in H2O, 80:10:10; from the 4th min a gradient was applied from
80:10:10 to 0:90:10 within 9 min, followed by 1 min of equilibration at 0:90:10 and 1 min at
80:10:10.
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1ꢀBenzylꢀ8ꢀmethoxyꢀ1H,3Hꢀpyrido[2,1ꢀf]purineꢀ2,4ꢀdione (5.)24 6ꢀAminoꢀ1ꢀbenzyluracil (4) 25
(10.8 g, 49.7 mmol, 1.00 eq.) was suspended in CH3CN (370 mL). Nꢀbromosuccinimide (17.7 g,
99.4 mmol, 2.00 eq.) was added to the suspension and the mixture was heated at 80 ⁰C for 1
hour, after which full conversion was shown by TLC (1:9 CH3OH/CH2Cl2 + 3% triethylamine).
Subsequently 4ꢀmethoxypyridine (15.1 mL, 149.2 mL, 3.00 eq.) was added and the mixture was
heated at 80 ⁰C during 10 hours. Full consumption of the bromo intermediate was shown by TLC
(1% CH3OH/CH2Cl2). A precipitate was formed overnight at RT which was collected by
filtration and washed with diethyl ether. This yielded the desired compound as a white solid
1
(10.2 g, 31.6 mmol, 64%). H NMR (400MHz, DMSOꢀd6) δ: 11.11 (s br, 1H), 8.72 (d, J = 7.2
Hz, 1H), 7.39ꢀ7.29 (m, 4H), 7.28ꢀ7.22 (m, 2H), 6.91 (dd, J = 7.2, 2.0 Hz, 1H), 5.19 (s, 2H), 3.89
(s, 3H) ppm. NMR was according to literature data.24
General procedure for the preparation of N3-substitited 1-benzyl-8-methoxy-1H,3H-
pyrido[2,1-f]purine-2,4-diones (1, 2, 6-22).24 The compounds were synthesized using the
procedure described by Priego et al.,24 but 5 eq. of the alkyl halide was used instead of 1.5 eq.
o
and the reaction mixture was heated to 80 C overnight in all cases. The pure compounds were
obtained by silica column chromatography using a mixture of petroleum ether/ethyl acetate (2:1)
as eluent, if not otherwise stated.
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1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀpropylꢀ1H,3Hꢀpyrido[2,1ꢀf]purineꢀ2,4ꢀdione (1).24 The pure product
was obtained by column chromatography using petroleum ether/ethyl acetate (1:1) as eluent,
yielding a white solid 113 mg, 0.31 mmol, 60%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2
Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.34ꢀ7.25 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6,
2.8 Hz, 1H), 5.37 (s, 2H), 4.02 (t, J = 7.6 Hz, 2H), 3.92 (s, 3H), 1.74 (sextet, J = 7.6 Hz, 2H),
0.99 (t, J = 7.6 Hz, 3H) ppm.24 MS [ESI+H]+: calcd for C20H20N4O3, 364.15; found, 365.0.
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1ꢀBenzylꢀ3ꢀ(cyclopropylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (2).23 The
pure product was obtained by column chromatography using a mixture of 2% CH3OH/CH2Cl2
as eluent, yielding a white solid, 1.51 g, 3.98 mmol, 86%. 1H NMR (400MHz, CDCl3) δ: 8.82 (d,
J = 7.2 Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.33ꢀ7.23 (m, 3H), 6.97 (d, J = 2.4 Hz, 1H), 6.73 (dd, J
= 8.4, 2.4 Hz, 1H), 5.37 (s, 2H), 3.94 (d, J = 7.4 Hz, 2H), 3.92 (s, 3H), 1.35ꢀ1.25 (m, 1H), 0.47ꢀ
0.44(m, 4H) ppm. MS [ESI+H]+: calcd for C21H20N4O3, 376.15; found, 376.9.
1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀmethylpyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (6).
Reaction was
performed in a sealed tube and 50 eq. of metyl iodide was used. The residue was purified by
silica column chromatography eluting with a petroleum ether/ethyl acetate (1:1) mixture,
1
yielding a white solid, 25 mg, 0.074 mmol, 15%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6
Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.33ꢀ7.24 (m, 3H), 6.99 (d, J = 2.4 Hz, 1H), 6.75 (dd, J = 7.6,
2.4 Hz, 1H), 5.37 (s, 2H), 3.93 (s, 3H), 3.45 (s, 3H) ppm. MS [ESI+H]+: calcd for C18H16N4O3,
336.12; found, 337.2.
1ꢀBenzylꢀ3ꢀethylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (7). The pure product was
obtained by column chromatography using petroleum ether/ethyl acetate (1:1) as eluent, yielding
1
a white solid, 58 mg, 0.17 mmol, 33%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H),
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7.54 (d, J = 7.2 Hz, 2H), 7.33ꢀ7.24 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6, 2.8 Hz,
1H), 5.36 (s, 2H), 4.12 (q, J = 7.2 Hz, 2H), 3.92 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H) ppm. MS
[ESI+H]+: calcd for C19H18N4O3, 350.14; found, 351.0.
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1ꢀBenzylꢀ3ꢀbutylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (8). Purified by column
chromatography using petroleum ether/EtOAc (3:1), yielding a white solid 10 mg, 0.026 mmol,
5%. 1H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 6.8 Hz, 2H), 7.24ꢀ7.33
(m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.4, 2.6 Hz, 1H), 5.36 (s, 2H), 4.04 (t, J = 7.6 Hz,
2H), 3.93 (s, 3H), 1.70ꢀ1.64 (m, 2H), 1.40 (sextet, J = 3.6 Hz, 2H), 0.95 (t, J = 7.2 Hz, 3H) ppm.
MS [ESI+H]+: calcd for C21H22N4O3, 378.17; found, 378.9.
1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀpentylꢀ1H,3Hꢀpyrido[2,1ꢀf]purineꢀ2,4ꢀdione (9). White solid, 110 mg,
1
0.28 mmol, yield 56%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 7.2
Hz, 2H), 7.34ꢀ7.25 (m, 3H), 6.97 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6, 2.8 Hz, 1H), 5.37 (s, 2H),
4.05 (t, J = 7.6 Hz, 2H), 3.93 (s, 3H), 1.72ꢀ1.66 (m, 2H), 1.39ꢀ1.37 (m, 4H), 0.91 (t, J = 7.2 Hz,
3H) ppm. MS [ESI+H]+: calcd for C22H24N4O3, 392.18; found, 393.1.
1ꢀBenzylꢀ3ꢀhexylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (10). White solid, 90 mg,
1
0.22 mmol, yield 44%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H), 7.55 (d, J = 6.8
Hz, 2H), 7.34ꢀ7.25 (m, 3H), 6.98 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz, 1H), 5.37 (s, 2H),
4.05 (t, J = 7.6 Hz, 2H), 3.92 (s, 3H), 1.69 (pentet, J = 7.6 Hz, 2H), 1.40ꢀ1.26 (m, 6H), 0.89 (t, J
= 7.2 Hz, 3H) ppm. MS [ESI+H]+: calcd for C23H26N4O3, 406.20; found, 407.1.
1ꢀBenzylꢀ3ꢀheptylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(11).
Column
chromatography using a mixture of petroleum ether/ethyl acetate (3:1) as eluent yielded 131 mg
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of the pure product as white solid, 0.31 mmol, 62%. 1H NMR (400MHz, CDCl3) H NMR
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(400MHz, CDCl3) δ: 8.80 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 6.8 Hz, 2H), 7.32ꢀ7.25 (m, 3H), 6.95
(d, J = 2.4 Hz, 1H), 6.71 (dd, J = 7.2, 2.4 Hz, 1H), 5.35 (s, 2H), 4.03 (t, J = 7.6, 2H), 3.90 (s,
3H), 1.67 (pentet, J = 7.6 Hz, 2H), 1.38ꢀ1.26 (m, 8H), 0.87 (t, J = 7.2 Hz, 3H) ppm. MS
[ESI+H]+: calcd for C24H48N4O3, 420.22; found, 421.2.
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3ꢀAllylꢀ1ꢀbenzylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (12). Purified by column
chromatography using petroleum ether/ethyl acetate (1:1) as eluent, yielding a white solid, 73
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mg, 0.20 mmol, 40%. H NMR (400MHz, CDCl3) δ: 8.82 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 7.2
Hz, 2H), 7.34ꢀ7.25 (m, 3H), 6.99 (d, J = 2.0 Hz, 1H), 6.75 (dd, J = 7.6, 2.4 Hz, 1H), 6.02ꢀ5.92
(m, 1H), 5.38 (s, 2H), 5.29 (dd, J = 17.2, 1.2 Hz, 1H), 5.20 (d, J = 10.0 Hz, 1H), 4.68 (d, J = 5.6
Hz, 2H), 3.93 (s, 3H) ppm. MS [ESI+H]+: calcd for C20H18N4O3, 362.14; found, 363.0.
1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀ(propꢀ2ꢀynꢀ1ꢀyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (13). The pure
product was obtained by column chromatography using petroleum ether/ethyl acetate (1:1) as
eluent, yielding a white solid, 60 mg, 0.17 mmol, 3%. 1H NMR (400MHz, CDCl3) δ: 8.82 (d, J =
7.2 Hz, 1H), 7.57 (d, J = 6.8 Hz, 2H), 7.36ꢀ7.26 (m, 3H), 6.99 (d, J = 2.4 Hz, 1H), 6.76 (dd, J =
7.2, 2.4 Hz, 1H), 5.38 (s, 2H), 4.83 (d, J = 2.4 Hz, 2H), 3.94 (s, 3H), 2.19 (t, J = 2.4 Hz, 1H)
ppm. MS [ESI+H]+: calcd for C20H16N4O3, 360.12; found, 361.1.
1ꢀBenzylꢀ3ꢀ(butꢀ3ꢀenꢀ1ꢀyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (14). White solid,
18 mg, 0.05 mmol, yield 10%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.6 Hz, 1H), 7.54 (d, J
= 7.2 Hz, 2H), 7.35ꢀ7.28 (m, 3H), 7.00 (d, J = 2.4 Hz, 1H), 6.77 (dd, J = 7.6, 2.4 Hz, 1H), 5.91ꢀ
5.84 (m, 1H), 5.39 (s, 2H), 5.08 (dd, J = 16.8, 1.2 Hz, 1H), 5.02 (d, J = 10.4, 1H), 4.15 (t, J = 7.2
Hz, 2H), 3.95 (s, 3H), 2.48 (q, J = 7.2 Hz, 2H) ppm. MS [ESI+H]+: calcd for C21H20N4O3,
376.15; found, 376.9.
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1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀ(2ꢀmethoxyethyl)ꢀ1H,3Hꢀpyrido[2,1ꢀf]purineꢀ2,4ꢀdione (15). Purified
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white solid, 70 mg, 0.18 mmol, 37%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H),
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7.54 (d, J = 6.8 Hz, 2H), 7.33ꢀ7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz,
1H), 5.36 (s, 2H), 4.30 (t, J = 6.0 Hz, 2H), 3.93 (s, 3H), 3.68 (t, J = 6.0 Hz, 2H), 3.36 (s, 3H)
ppm. MS [ESI+H]+: calcd for C20H20N4O3, 380.15; found, 380.8.
1ꢀBenzylꢀ3ꢀ(3ꢀhydroxypropyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (16). Purified
by column chromatography using a mixture of petroleum ether/EtOAc (1:2), followed by
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recrystallization from CH3OH/EtOAc. Yield: white solid, 106 mg, 0.28 mmol, 28%. H NMR
(400MHz, CDCl3) δ: 8.80 (d, J = 7.2 Hz, 1H), 7.53 (d, J = 7.6 Hz, 2H), 7.33ꢀ7.24 (m, 3H), 6.99
(d, J = 2.4 Hz, 1H), 6.76 (dd, J = 7.6, 2.4 Hz, 1H), 5.37 (s, 2H), 4.22 (t, J = 6.0 Hz, 2H), 3.93 (s,
3H), 3.53 (t, J = 5.6 Hz, 2H), 1.92 (pentet, J = 5.6 Hz, 2H) ppm. MS [ESI+H]+: calcd for
C20H20N4O3, 380.15; found, 380.9.
1ꢀBenzylꢀ3ꢀisobutylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (17). White solid, 0.08
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mmol, 29 mg, yield 15%. H NMR (400MHz, CDCl3) δ: 8.86 (d, J = 7.2 Hz, 1H), 7.55 (d, J =
7.2 Hz, 2H), 7.33ꢀ7.28 (m, 3H), 7.00 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 7.6, 2.4 Hz, 1H), 5.39 (s,
2H), 3.95 (s, 3H), 3.92 (d, J = 7.6 Hz, 2H), 2.22 (nonet, J = 7.2 Hz, 1H), 0.97 (d, J = 6.8 Hz, 6H)
ppm. MS [ESI+H]+: calcd for C21H22N4O3, 378.17; found, 379.0.
1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀ(3ꢀneopentyl)ꢀ1H,3Hꢀpyrido[2,1ꢀf]purineꢀ2,4ꢀdione (18). Purified by
column chromatography using a mixture of 1% CH3OH in CH2Cl2 as eluent, yielding the product
as a white solid, 0.025 mmol, 20mg, 5%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.2 Hz, 1H),
7.52 (d, J = 7.2 Hz, 2H), 7.32ꢀ7.25 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 7.2, 2.4 Hz,
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1H), 5.37 (s, 2H), 3.99 (s, 2H), 3.92 (s, 3H), 0.98 (s, 9H) ppm. MS [ESI+H]+: calcd for
C22H24N4O3, 392.18; found, 393.0.
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1ꢀBenzylꢀ3ꢀisopentylꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (19). Yield: white solid,
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104 mg, 0.26 mmol, 53%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J = 7.2 Hz, 1H), 7.53 (d, J =
7.2 Hz, 2H), 7.33ꢀ7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz, 1H), 5.36 (s,
2H), 4.08ꢀ4.04 (m, 2H), 3.93 (s, 3H), 1.69 (nonet, J = 6.8 Hz, 1H), 1.59ꢀ1.53 (m, 2H), 0.98 (d, J
= 6.4 Hz, 6H) ppm. MS [ESI+H]+: calcd for C22H24N4O3, 392.18; found, 393.1.
1ꢀBenzylꢀ3ꢀ(3,3ꢀdimethylbutyl)ꢀ8ꢀmethoxyꢀpyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (20). Yield:
white solid, 81 mg, 0.20 mmol, 40%. 1NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.6 Hz, 1H), 7.55
(d, J = 7.2, 2H), 7.35ꢀ7.27 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 7.2, 2.4 Hz, 1H), 5.38
(s, 2H), 4.12ꢀ4.08 (m, 2H), 3.93 (s, 3H), 1.61ꢀ1.57 (m, 2H), 1.04 (s, 9H) ppm. MS [ESI+H]+:
calcd for C23H26N4O3, 406.20; found, 407.1.
1ꢀBenzylꢀ8ꢀmethoxyꢀ3ꢀ((trimethylsilyl)methyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(21).
White solid, 137 mg, 0.34 mmol, yield 67%. 1H NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.2 Hz,
1H), 7.50 (d, J = 6.8 Hz, 2H), 7.32ꢀ7.24 (m, 3H), 6.98 (d, J = 2.4 Hz, 1H), 6.73 (d, J = 7.2, 2.4
Hz, 1H), 5.39 (s, 2H), 3.93 (s, 3H), 3.64 (s, 2H), 0.08 (s, 9H) ppm. MS [ESI+H]+: calcd for
C21H24N4O3Si, 408.16; found, 409.2.
1ꢀBenzylꢀ3ꢀ(cyclobutylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (22). White
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solid, 90 mg, 0.23 mmol, yield 46%. H NMR (400MHz, CDCl3) δ: 8.82 (d, J = 7.2 Hz, 1H),
7.53 (d, J = 7.2 Hz, 2H), 7.33ꢀ7.13 (m, 3H), 6.69 (d, J = 1.8 Hz, 1H), 6.73 (dd, J = 7.2, 2.4 Hz,
1H), 5.35 (s, 2H), 4.11 (d, J = 7.6 Hz, 2H), 3.92 (s, 3H), 2.83ꢀ2.72 (m, 1H), 2.05ꢀ1.95 (m, 2H),
1.90ꢀ1.79 (m, 4H) ppm. MS [ESI+H]+: calcd for C22H22N4O3, 390.17; found, 391.2.
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3ꢀ(Cyclopropylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (23).23 Prepared
following a slightly modified procedure described by Priego et al. In total 4 portions of 8 eq. of
ammonium formate (after 0, 2, 4 and 6 hours) and 3 portions of 0.15 eq. of 20% Pd(OH)2 (0, 4
and 6 hours) were added, after which full conversion was reached after overnight reflux
visualized by TLC (3% CH3OH/CH2Cl2). The reaction mixture was filtered over Celite and the
residue extracted 5 times with hot DMF. The combined organic layer was concentrated in vacuo
which resulted in a quantitative yield. 1HꢀNMR in accordance to data in literature.23
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General procedure for the preparation of N1-substitited-3-cyclopropylmethyl-8-methoxy-
1H,3H-pyrido[2,1-f]purine-2,4-diones (24-33).23 The compounds were synthesized according
to the procedure described by Priego et al.2 Purification by silica column chromatography using
an eluent mixture of petroleum ether/ethyl acetate (3:1) yielded the pure final products.
3ꢀ(Cyclopropylmethyl)ꢀ8ꢀmethoxyꢀ1ꢀ(3ꢀmethylbenzyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
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(24). White solid, 79 mg, 0.20 mmol, yield 57%. H NMR (400MHz, CDCl3) δ: 8.84 (d, J = 7.2
Hz, 1H), 7.36 (s, 2H), 7.22 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H),
6.75 (dd, J = 7.2, 2.4 Hz, 1H), 5.36 (s, 2H), 3.97 (d, J = 7.2 Hz, 2H), 3.94 (s, 3H), 2.34 (s, 3H),
1.37ꢀ1.31 (m, 1H), 0.52ꢀ0.45 (m, 4H) ppm. MS [ESI+H]+: calcd for C22H22N4O3, 390.17; found,
391.0.
3ꢀ(Cyclopropylmethyl)ꢀ8ꢀmethoxyꢀ1ꢀ(4ꢀmethylbenzyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(25).23
3ꢀ(Cyclopropylmethyl)ꢀ1ꢀ(4ꢀethylbenzyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
1
(26). White solid, 43 mg, 0.11 mmol, yield 21%. H NMR (400MHz, CDCl3) δ: 8.82 (d, J = 7.6
Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 2.0 Hz, 1H), 6.73 (dd, J =
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7.2, 2.4 Hz, 1H), 5.34 (s, 2H), 3.95ꢀ3.93 (m, 5H), 2.60 (q, J = 7.6 Hz, 2H), 1.34ꢀ1.28 (m, 1H),
1.19 (t, J = 7.6 Hz, 3H), 0.50ꢀ0.42 (m, 4H) ppm. MS [ESI+H]+: calcd for C23H24N4O3, 404.18;
found, 405.2.
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3ꢀ(Cyclopropylmethyl) ꢀ8ꢀmethoxyꢀ1ꢀ(3ꢀmethoxybenzyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
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(27). White solid, 20 mg, 0.049 mmol, yield 14%. H NMR (400MHz, CDCl3) δ: 8.83 (d, J =
7.6 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.15ꢀ7.10 (m, 2H), 6.97 (d, J = 1.6 Hz, 1H), 6.80 (dd, J =
8.4, 2.4 Hz, 1H), 6.74 (dd, J = 7.6, 2.4 Hz, 1H), 5.35 (s, 2H), 3.96ꢀ3.92 (m, 5H), 3.77 (s, 3H),
1.35ꢀ1.28 (m, 1H), 0.50ꢀ0.45 (m, 4H) ppm. MS [ESI+H]+: calcd for C22H22N4O4, 406.16; found,
406.9.
3ꢀ(Cyclopropylmethyl)ꢀ8ꢀmethoxyꢀ1ꢀ(4ꢀmethoxybenzyl)pyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(28).23
1ꢀ(3ꢀChlorobenzyl)ꢀ3ꢀ(cyclopropylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(29).23
1ꢀ(4ꢀChlorobenzyl)ꢀ3ꢀ(cyclopropylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
(30).23
3ꢀ(Cyclopropylmethyl)ꢀ1ꢀ(3,4ꢀdichlorobenzyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀ
dione (31).23
1ꢀ(4ꢀBromobenzyl)ꢀ3ꢀ(cyclopropylmethyl)ꢀ8ꢀmethoxypyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione
1
(32). White solid, 22 mg, 0.048 mmol, yield 14%. H NMR (400MHz, CDCl3) δ: 8.85 (d, J =
7.2 Hz, 1H), 7.46 (s, 4H), 6.99 (d, J = 1.6 Hz, 1H), 6.87 (dd, J = 7.2, 2.0 Hz, 1H), 5.33 (s, 2H),
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3.99ꢀ3.85 (m, 5H), 1.34ꢀ1.24 (m, 1H), 0.50ꢀ0.44 (m, 4H) ppm. MS [ESI+H]+: calcd for
C21H19N4O3, 454.06; found, 455.0.
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3ꢀ(Cyclopropylmethyl) ꢀ8ꢀmethoxyꢀ1ꢀphenylethylpyrido[2,1ꢀf]purineꢀ2,4(1H,3H)ꢀdione (33).
White solid, 112 mg, 0.29 mmol, yield 82%. 1H NMR (400MHz, CDCl3) δ: 8.81 (d, J = 7.2 Hz,
1H), 7.35ꢀ7.25 (m, 4H), 7.20 (t, J = 7.2 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.73 (dd, J = 7.2, 2.4
Hz, 1H), 4.40 (t, J = 8.0 Hz, 2H), 3.95ꢀ3.90 (m, 5H), 3.12 (t, J = 8.0 Hz, 2H), 1.32ꢀ1.23 (m, 1H),
0.49ꢀ0.40 (m, 4H) ppm. MS [ESI+H]+: calcd for C22H22N4O3, 390.17; found, 391.0.
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Biology. Chemicals and Reagents. [3H]8ꢀEthylꢀ4ꢀmethylꢀ2ꢀphenylꢀ(8R)ꢀ4,5,7,8ꢀtetrahydroꢀ
1Hꢀ imidazo[2,1ꢀi]ꢀpurinꢀ5ꢀone26 ([3H]34, specific activity 56 Ci·mmolꢀ1) was a gift from Prof.
C.E. Müller (University of Bonn, Germany). Unlabeled 34 was purchased from Tocris Ltd.
(Abingdon, UK). 5’ꢀNꢀethylcarboxamidoadenosine (NECA) was purchased from SigmaꢀAldrich
(Steinheim, Germany). Adenosine deaminase (ADA) was purchased from Boehringer Mannheim
(Mannheim, Germany). Bicinchoninic acid (BCA) and BCA protein assay reagents were
purchased from Pierce Chemical Company (Rockford, IL, USA). Chinese hamster ovary cells
stably expressing the human adenosine A3 receptor (CHOhA3) were a gift from Dr. KꢀN Klotz
(University of Würzburg, Germany). All other chemicals were obtained from standard
commercial sources and were of analytical grade.
Cell Culture and Membrane Preparation. Chinese Hamster Ovary (CHO) cells, stably
expressing the human adenosine A3 receptor (CHOhA3), were cultured and membranes were
prepared and stored as previously described44. Protein determination was done through use of the
bicinchoninic acid (BCA) method45.
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Radioligand Displacement Assay. Membrane aliquots containing ~15 ꢁg of CHOhA3 protein
were incubated in a total volume of 100 ꢁL assay buffer (50 mM TrisꢀHCl, 5 mM MgCl2,
supplemented with 0.01% CHAPS and 1 mM EDTA, pH 7.4) at 25 °C for 120 min.
Displacement experiments were performed using 6 concentrations of competing antagonist in the
presence of a final concentration of ~10 nM [3H] 34. At this concentration, total radioligand
binding did not exceed 10% of that added to prevent ligand depletion. Nonspecific binding
(NSB) was determined in the presence of 100 ꢁM NECA. Incubation was terminated by rapid
filtration performed on 96ꢀwell GF/B filter plates (Perkin Elmer, Groningen, the Netherlands),
using a PerkinElmer Filtermateꢀharvester (Perkin Elmer, Groningen, the Netherlands). After
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drying the filter plate at 50 C for 30 min, the filterꢀbound radioactivity was determined by
scintillation spectrometry using the 2450 MicroBeta2 Plate Counter (Perkin Elmer, Boston, MA).
Radioligand association and dissociation assays. Association experiments were performed by
incubating membrane aliquots containing ~15 ꢁg of CHOhA3 membrane in a total volume of 100
ꢁL of assay buffer at 10 or 25 °C with ~10 nM [3H] 34. The amount of radioligand bound to the
receptor was measured at different time intervals during a total incubation of 120 min.
Dissociation experiments were performed by preincubating membrane aliquots containing ~15
ꢁg of protein in a total volume of 100 ꢁL of assay buffer at 10 or 25 °C for 60 min. After the
preincubation, radioligand dissociation was initiated by the addition of 5 ꢁl 100 ꢁM unlabeled
NECA. The amount of radioligand still bound to the receptor was measured at various time
intervals for a total of 120 min to ensure that full dissociation from hA3 receptor was reached.
Incubations were terminated and samples were obtained as described under Radioligand
Displacement Assay.
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Radioligand Competition Association Assay. The binding kinetics of unlabeled ligands were
quantified using the competition association assay based on the theoretical framework by
Motulsky and Mahan.46 The competition association assay was initiated by adding membrane
aliquots (15 ꢁg/well) at different time points for a total of 240 min to a total volume of 100 ꢁl of
assay buffer at 10 °C or 25 °C with ~10 nM [3H] 34 in the absence or presence of a single
concentration of competing hA3R antagonists (i.e. at their IC50 value). Incubations were
terminated and samples were obtained as described under Radioligand Displacement Assay. The
“dualꢀpoint” competition association assays were designed as described previously,27 where in
this case the two time points were selected at 20 (t1) and 240 min (t2).
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[35S] GTPγS Binding Assay. The assays were performed by incubating 15 µg of homogenized
CHOhA3 membranes in a total volume of 80 µl assay buffer (50 mM TrisꢀHCl buffer, 5 mM
MgCl2, 1 mM EDTA, 0.05% BSA and 1 mM DTT, pH 7.4) supplemented with 1 µM GDP and 5
µg saponin. The assays were performed in a 96ꢀwell plate format, where DMSO stock solutions
of the compounds were added using a HP D300 Digital Dispenser (Tecan, Männedorf,
Switserland). The final concentration of organic solvent per assay point was ≤0.1%. In all cases,
the basal level of [35S] GTPγS binding was measured in untreated membrane samples, whereas
the maximal level of [35S] GTPγS binding was measured by treatment of the membranes with 10
ꢁM 2ꢀClꢀIBMECA. For the insurmountability experiments, membrane preparations were preꢀ
o
incubated with or without antagonists (30ꢀ, 100ꢀ, 300ꢀfold Ki values) for 60 min at 25 C, prior
to the addition of 2ꢀClꢀIBMECA (10 µM to 0.1 nM) and 20 µl [35S] GTPγS (final concentration
~0.3 nM), after which incubation continued for another 30 min at 25 oC. For the surmountability
(control) experiments, antagonists and 2ꢀClꢀIBMECA were coꢀincubated with [35S] GTPγS for
30 min at 25 oC. For all experiments, incubations were terminated and samples were obtained as
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described under Radioligand Displacement Assay, by using GF/B filters (Whatman International,
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Maidstone, UK).
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Data Analysis. All experimental data were analyzed using the nonlinear regression curve
fitting program GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA). From
displacement assays, IC50 values were obtained by nonꢀlinear regression analysis of the
displacement curves. The obtained IC50 values were converted into Ki values using the Chengꢀ
Prusoff equation to determine the affinity of the ligands.47 The observed association rates (kobs)
derived from both assays were obtained by fitting association data using one phase exponential
association. The dissociation rates were obtained by fitting dissociation data to a one phase
exponential decay model. The kobs values were converted into association rate constants (kon)
using the equation kon = (kobs – koff)/[L], where [L] is the amount of radioligand used for the
association experiments. The association and dissociation rates were used to calculate the kinetic
KD using the equation KD = koff/kon. Association and dissociation rate constants for unlabeled
compounds were calculated by fitting the data into the competition association model using
“kinetics of competitive binding”:46
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K A = k1[L]⋅10−9 + k2
KB = k3[I]⋅10−9 + k4
S = (K A − KB )2 + 4 ⋅ k1 ⋅ k3 ⋅ L ⋅ I ⋅10−18
KF = 0.5(K A + KB + S)
KS = 0.5(K A + KB − S)
Bmax ⋅ k1 ⋅ L ⋅10−9
Q =
KF − KS
k4 ⋅(KF − KS ) k4 − KF
k4 − KS
F ⋅X )
S ⋅X )
Y = Q ⋅ (
+
e(−K
−
e(−K
)
KF ⋅ KS
KF
KS
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