Exploring the directionality of 5-substitutions in a new series of 5-alkylaminopyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine as a strategy to design novel human A3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm300899q

The structure-activity relationship (SAR) of new 5-alkylaminopyrazolo[4,3- e]1,2,4-triazolo[1,5-c]pyrimidines as antagonists of the A₃ adenosine receptor (AR) was explored with the principal aim to establish the directionality of 5-substitutions inside the orthosteric binding site of the A₃ AR. All the synthesized compounds showed affinity for the hA ₃ AR from nanomolar to subnanomolar range. In particular, the most potent and selective antagonist presents an (S) α-phenylethylamino moiety at the 5 position (26, K_i hA₃ = 0.3 nM). Using an in silico receptor-driven approach, we have determined the most favorable orientation of the substitutions at the 5 position of the pyrazolo[4,3-e]1,2,4- triazolo[1,5-c]pyrimidine (PTP) scaffold, opening the possibility for further derivatizations aimed at directing the N⁵ position toward the extracellular environment.

Full text of DOI:10.1021/jm300899q

Article
pubs.acs.org/jmc
Exploring the Directionality of 5‑Substitutions in a New Series
of 5‑Alkylaminopyrazolo[4,3‑e]1,2,4-triazolo[1,5‑c]pyrimidine
as a Strategy To Design Novel Human A3 Adenosine
Receptor Antagonists.
Stephanie Federico,† Antonella Ciancetta,‡ Davide Sabbadin,‡ Silvia Paoletta,‡ Giorgia Pastorin,§
Barbara Cacciari,⊥ Karl Norbert Klotz,□ Stefano Moro,*,‡ and Giampiero Spalluto*,†
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
̀
di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
‡Molecular Modeling Section (MMS), Dipartimento di Scienze del Farmaco, Universita
I-35131 Padova, Italy
̀
di Padova, via Marzolo 5,
§Department of Pharmacy, National University of Singapore, 3 Science Drive 2, Singapore 117543
⊥Dipartimento di Scienze Farmaceutiche, Universita
□Institut fur Pharmakologie, Universitat of Wurzburg, D-97078 Wurzburg, Germany
̀
degli Studi di Ferrara, via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
̈
̈
̈
̈
S
* Supporting Information
ABSTRACT: The structure−activity relationship (SAR) of new 5-alkylaminopyrazolo-
[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines as antagonists of the A3 adenosine receptor
(AR) was explored with the principal aim to establish the directionality of
5-substitutions inside the orthosteric binding site of the A3 AR. All the synthesized
compounds showed affinity for the hA3 AR from nanomolar to subnanomolar
range. In particular, the most potent and selective antagonist presents an (S)
α-phenylethylamino moiety at the 5 position (26, Ki hA3 = 0.3 nM). Using an in
silico receptor-driven approach, we have determined the most favorable orientation
of the substitutions at the 5 position of the pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]-
pyrimidine (PTP) scaffold, opening the possibility for further derivatizations aimed
at directing the N5 position toward the extracellular environment.
antagonists as potential antiasthmatic,19 antinflammatory, and
INTRODUCTION
■
cerebroprotective agents and for the treatment of glaucoma.9,20,21
For this purpose, in the past few years, different classes of
heterocyclic derivatives have emerged as A3 AR antagonists.22
In this research field, our group has reported a large series of
pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine (PTP) derivatives
as highly potent and selective human A3 adenosine receptor
(hA3 AR) antagonists.23−28 In particular, we observed that the
simultaneous introduction of a methyl group at the N8 position
and the presence of aryl carbamoyl (1) or arylacetyl (2) moieties
at the N5 position led to potent and selective hA3 AR antagonists
(Chart 1).23−25
Adenosine is a neuromodulator exerting several functions
through the activation of specific G protein-coupled receptors
that are classified into four different subtypes, termed A1, A2A,
A2B, and A3, based on pharmacological criteria and coupling to
adenylyl cyclase.1,2 The adenosine A3 receptor (A3 AR) was first
cloned from a rat testis cDNA library3 and is still undergoing
intensive pharmacological characterization. Subsequently, A3
ARs have also been cloned from several other species such as
human,4,5 sheep,6 mouse,7 and rabbit.8 However, significant
differences in sequence similarity (72%) have been detected
among different species:9,10 The rat A3 adenosine receptor (rA3 AR),
especially, behaves anomalously in ligand binding assays.11
The activation of A3 ARs is related to various second
messenger systems: In particular, they have been shown to
stimulate phospholipase C12 and D13 and to inhibit adenylyl
cyclase.14 In the rat, the activation of A3 ARs results in hypo-
tension through the promotion of the release of inflammatory
mediators from mast cells.15 It has also been suggested that
the A3 AR plays an important role in brain ischemia,16
immunosuppression,17 and cellular growth.18
In addition, we have recently reported a novel series of
PTP-based fluorophore-conjugated hA3 AR antagonists with a
versatile application in cell-based assay ligand discovery and
receptor localization imaging.29 Herein, we report the essential
pilot work based on an extensive exploration of new possible
spacers at the N5 position as a site of linkage not only for
fluorophore groups but also for other possible functionalizing
moieties that can be useful in the study of the physiological and
On the basis of these pharmacological observations, many
Received: June 26, 2012
efforts have been made in search of highly selective A3 AR
© XXXX American Chemical Society
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Chart 1. Arylcarbamoyl and Arylacetyl Pyrazolo-triazolo-
pyrimidine Derivatives as Human A3 Adenosine Receptor
Antagonists
RESULTS AND DISCUSSION
■
A. Chemistry. All the designed compounds 3−62 have
been synthesized as summarized in Scheme 1. By reacting the
well-known triazolylpyrazolamino derivative 6324 with diphos-
gene in dry dioxane, the corresponding cyclized derivative 64
was obtained. The latter, after treatment with a mixture of
POCl3/PCl5 in pyridine, afforded the chloro derivative 65
which, after reaction with sodium ethoxide or methoxide in dry
ethanol or methanol at reflux, gave the corresponding alkyloxy
derivatives 3 and 4. Instead, by reacting the chloro derivative 65
with an appropriate alkyl- or aralkylamine in ethanol in a sealed
tube at 110 °C, the corresponding 5-(ar)alkylamino derivatives
5−62 were obtained.
physiopathological roles of ARs. We focused on substituents of
different natures and sizes, to evaluate their ability to direct the
N5 position toward the extracellular environment.
To this aim, we decided to modify the synthetic approach to
the PTP nucleus (see Chemistry): The new approach has
enabled us to introduce a large variety of polyfunctionalized
chains, such as alkoxy or alkyl amino moieties, at the 5 position
under mild conditions, leading to the design and synthesis of
compounds 3−62 (Chart 2). Along with the pharmacological
B. Biological Activity. The newly synthesized compounds
(3−62, 64, 65) were tested at the human A1, A2A, and A3
receptors expressed in CHO cells: [3H]CCPA (A1) and
[3H]NECA (A2A, A3) were used as radioligands in binding
assays.30 As far as the activity at the human A2B AR is concerned,
the inhibition of NECA-stimulated adenylyl cyclase activity was
determined as a measurement of the affinity of the compounds.
Structure−Activity Relationships. As clearly summarized in
Table 1, all the synthesized compounds (3−62, 64, 65) showed
affinities at the hA3 AR ranging from high nanomolar to sub-
nanomolar concentrations, with different degree of selectivity
versus hA1 AR and hA2A AR, while being almost inactive at the
hA2B AR. It is quite evident that compounds lacking the amino
group at the 5 position (3, 64, 65) exhibit low affinity at the
hA3 AR. An exception is represented by compound 4 bearing
an ethoxy moiety at the 5 position, which possesses quite good
affinity (hA3 AR Ki = 182 nM) but low levels of selectivity
versus the other receptor subtypes.
Chart 2. General Structures of Synthesized Compounds 3−62
evaluation, the new derivatives have also been subjected to
molecular modeling studies, to better analyze the essential
structural requirements to direct the N5 position toward the
extracellular environment, while retaining both affinity and
selectivity at the hA3 AR. Therefore, the new derivatives herein
reported may help in the design and development of new
potent hA3 AR antagonists as well as linkage points for
functionalizing groups aimed at the study of ARs functions.
A significant recovery of the affinity at the hA3 AR can be
observed with the 5-alkylamino derivatives (5−62). A detailed
analysis of binding data has clearly revealed the effect of the
substituent at the N5 position on the hA3 AR affinity and
selectivity. In particular, small alkyl groups such as methyl (5)
or ethyl (6) give pretty good affinity at the hA3 AR (hA3 AR
Ki = 63.3 nM and hA3 AR Ki = 44.4 nM, respectively), but the
a
Scheme 1
a
Reagents and conditions: (i) Diphosgene, dry dioxane, 70 °C, 90 min; (ii) POCl3/PCl5, dry pyridine, reflux, 24 h; (iii) RONa, ROH, reflux, 2 h;
(iv) R1RNH, EtOH, 110 °C, sealed tube, 2 h.
B
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levels of selectivity are still poor. Interestingly, elongation of the
chain at the N5 position increases the affinity (e.g., compound
11, hA3 AR Ki = 14.6 nM) but without selectivity versus the
other receptor subtypes.
5 position: the (S) isomer (26) is 10-fold more potent at the
hA3 AR than its (R) enantiomer (27) (26: hA3 AR Ki = 0.3 nM;
27: hA3 AR Ki = 3.7 nM) with an improved selectivity as a
consequence (26: hA1/hA3 = 1127; hA2A/hA3 = 184; 27: hA1/
hA3 = 255; hA2A/ hA3 = 10.3). It should be otherwise noted that
a similar effect of the stereochemistry has not been observed for
derivatives 9 and 10, suggesting that also the kind of substitution
plays a fundamental role in the receptor recognition.
Further elongation of the spacer between the phenyl ring and
the amino function (37, 38) or its replacement with a hetero-
cycle such as indole (39) significantly decreases the affinity and
the selectivity at the hA3 AR.
Further support that the increase of the liphophilic character
might help in achieving activity at the hA3 AR is suggested by
the binding data of compounds 40−50: In fact, the introduc-
tion of polar chains bearing hydroxyl functions at the N5
position is detrimental for both the affinity and the selectivity
(e.g., compound 44: hA3 AR Ki = 536 nM; hA1/hA3 = 1.2;
hA2A/hA3 = 1.1), and only when the aliphatic chain is longer
and/or branched is a partial recovery of the affinity observed,
even though the selectivity still remains poor (e.g., compound
43: hA3 AR Ki = 62.9 nM; hA1/hA3 = 3.5; hA2A/hA3 = 3.3).
Moreover, etherification of the hydroxyl group permits, in
part, affinity at the hA3 AR but without significant selectivity.
In particular, derivative 50, bearing at the 5 position a
phenoxyethyl amino group, shows quite good affinity at the
hA3 AR (Ki = 38.6 nM) but with low levels of selectivity versus
the other receptor subtypes (hA1/hA3 = 16.7; hA2A/hA3 =
3.13). Disubstitutions of the nitrogen at the 5 position with
small alkyl groups such as methyl (51) or ethyl (53) leads to
a significant reduction of the affinity with consequent loss of
selectivity (e.g., compound 51: hA3 AR Ki = 394 nM; hA1/
hA3 = 12.1; hA2A/hA3 = 2.61). Nevertheless, if one of the
substituents is a bulky group, such as n-butyl (52) or benzyl
(56), an increase of both affinity and selectivity is observed
(e.g., compound 56: hA3 AR Ki = 18 nM; hA1/hA3 = 31.4;
hA2A/hA3 = 60), while the simultaneous introduction of two
bulky substituents (e.g., compound 57: hA3 AR Ki = 404 nM;
hA1/hA3 = 1.8; hA2A/hA3 = 2.9) or a presence of hydrophilic
functions (e.g., compound 58: hA3 AR Ki = 1,360 nM; hA1/
hA3 = 11; hA2A/hA3 = 4.7) dramatically reduces the affinity and
the selectivity at the hA3 AR.
Introduction of cyclic amines at the 5 position produces
different effects on the binding profile depending on the nature
of the heterocycle. While pyrrolidine (59) or piperidine (60)
are well tolerated in terms of affinity (e.g., compound 59: hA3
AR Ki = 35.5 nM), the introduction of heterocycles with a
protonable basic moiety at the 4 position such as piperazine
(61) or morpholine (62) leads to a drastic reduction of the
affinity at the hA3 AR (e.g., compound 61: hA3 AR Ki = 1330 nM).
Nevertheless, for all these derivatives (59−62), the selectivity
versus the other receptor subtypes is very low.
C. Molecular Modeling. With the aim to rationalize from a
molecular point of view the observed binding data, all the newly
synthesized analogues have been subjected to a molecular
modeling investigation. The new derivatives were docked into
the binding cavity of the hA3 AR with the aim to identify their
hypothetical binding mode at this receptor subtype by focusing
the attention on the orientation of the N5 substituents. As,
to date, no crystallographic information about the A3 AR is
available, we performed the docking studies by using a hA3
AR homology model, which has been previously proposed31
and built by using the crystal structure of the hA2A AR32 as
Branched chains such as sec-butyl (8) also significantly
increase the affinity at the hA3 AR with an acceptable selectivity
(hA1/hA3 = 25; hA2A/hA3 = 47). By implementing the
lipophilic character of the chain at the N5 position (e.g.,
compound 14), both affinity and selectivity are significantly
increased (hA3 AR Ki = 3.82 nM; hA1/hA3 = 158; hA2A/hA3 =
134), whereas the introduction of bulky substituents (e.g.,
compound 15) reduces both the affinity and the selectivity
(hA3 AR Ki = 130 nM; hA1/hA3 = 7.5; hA2A/hA3 = 25).
A similar behavior can be observed with the introduction of
cycloalkyl substituents at the N5 position. In general, all the
cycloalkyl amino derivatives (16−22) show good affinity at
the hA3 AR. In particular, a good affinity can be obtained by
introducing five- (18 hA3 AR Ki = 7.68 nM) or six-membered
rings (19 hA3 AR Ki = 8.57 nM), while smaller (e.g., 16 hA3 AR
Ki = 69.3 nM) or larger (e.g., 20 hA3 AR Ki = 15.4 nM) rings
lead to a significant reduction of the affinity. However, bulky
substituents, such as the c-octyl (21) and 2-adamantyl (24)
moieties, are more tolerated than the smaller cyclopropyl (16)
group (21 hA3 AR Ki =15.5 nM, 24 hA3 AR Ki =18.8 nM vs
16 hA3 AR Ki =69.3 nM). Also the selectivity versus the
other receptor subtypes increases from the cyclopropyl (16:
hA1/hA3 = 3; hA2A/hA3 = 1.63) to the cyclohexyl (19: hA1/hA3 =
19.4; hA2A/hA3 = 27.2) derivatives, but generally the levels of
selectivity are very low.
In the cycloalkylamino series, the two adamantyl derivatives
23 and 24 show completely different binding profiles: In fact,
while the 2-isomer (24) is more potent at the hA3 AR (hA3
AR Ki = 18.8 nM vs hA3 AR Ki = 37.8 nM for 24 and 23,
respectively), the selectivity profile versus the other receptor
subtypes is the exact opposite (23: hA1/hA3 = 87; hA2A/hA3 =
>794; 24: hA1/hA3 = 39.9; hA2A/hA3 = 45.1).
Introduction of a benzyl group at the N5 position (25) leads
to an improvement of the affinity at the hA3 AR with good
levels of selectivity (hA3 AR Ki = 2.5 nM; hA1/hA3 = 212;
hA2A/hA3 = 36.5). Substitutions on the phenyl ring seems not
to have significant effects on both the hA3 AR affinity and
selectivity, with the exception of the 4-methyl derivative (28),
which displays increased affinity and selectivity (hA3 AR
Ki = 1.07 nM; hA1/hA3 = 503; hA2A/hA3 = 109). Conversely, the
presence of a 4-CF3 (32), 4-Ph (34), or phenyl disubstitutions
(33) is detrimental in terms of affinity and selectivity (e.g., com-
pound 34 hA3 AR Ki = 187 nM; hA1/hA3 = 93; hA2A/hA3 = 3).
Substitution at the N5 position with a β-phenylethyl moiety
(36) also leads to a significant loss of affinity and selectivity
(hA3 AR Ki = 52.8 nM; hA1/hA3 = 13; hA2A/hA3 = 0.8), thus
confirming that the presence of a carbonyl group such as in the
reference compound 2 plays a fundamental role in the receptor
recognition as previously reported.22−27 Nevertheless, the data
reported in Table 1 suggest that the introduction of additional
hydrophobic groups might balance the carbonyl lacking and
allow the recovery of the affinity. In fact, the benzhydryl
derivative 35 shows a Ki value at the hA3 AR of 0.83 nM and
high levels of selectivity versus the other receptor subtypes
(hA1/hA3 = 204; hA2A/hA3 = 1084). Also with an α-phenylethyl
moiety at the N5 position, a very potent and selective compound
is obtained (26: hA3 AR Ki = 0.3 nM; hA1/hA3 = 1,127; hA2A/
hA3 = 184). Considering the α-phenylethyl substitution, it is
quite evident the effect of the stereochemistry of the chain at the
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Figure 1. (A) Per residue electrostatic interaction energy map and (B) per residue hydrophobic interaction score map. The maps are calculated for a
selected pose of each compound (3−62) inside the hA3 AR binding site. Electrostatic energy values are expressed in kcal mol−1, whereas
hydrophobic scores are expressed in arbitrary hydrophobic units.
template. Moreover, docking simulations at the hA2A AR binding
site were also performed to explain the hA2A/hA3 selectivity
profile.
analogues, with a few exceptions. Asn250 (6.55) in TM6 is
highly conserved among all AR subtypes and believed to play
a role in ligand binding to the hA3 AR.33,34 On the other hand,
the map of the hydrophobic IEFs (Figure 1B) highlights several
residues involved in hydrophobic contacts with the ligands,
such as Leu68 (2.60), Val72 (2.64) in TM2, Leu90 (3.31) and
Leu91 (3.32) in TM3, Phe168 and Val169 in EL2, Trp243
(6.48), Leu246 (6.51) and Ile253 (6.58) in TM6, and Ile268
(7.39) in TM7.
The same interaction patterns have also been observed for
the energetically more favorable pose of the ligands at the
hA2A AR (Figure S1, Supporting Information): In particular,
the compounds establish electrostatic interactions with Asn253
(6.55) and Glu169 (EL2) and hydrophobic contacts with, among
others, Ile66 (2.64), Val84 (3.32), Phe168 (EL2), Leu249 (6.51),
and Ile274 (7.39). Consistently with their lower activity at the
hA2A AR, the per residue contributions are weaker with respect to
those calculated for the interactions with the hA3 AR residues.
The general trend that emerged from the above-described maps
has also been confirmed by a detailed analysis of the docking
poses, as reported below.
In addition, to analyze the ligand−receptor recognition
mechanism in a more quantitative fashion, we calculated the
individual electrostatic and hydrophobic contributions to the
interaction energy of each receptor residue involved in the
binding with the ligands. The analysis of these contributions
afforded “interaction energy fingerprints” (hereby indicated as
IEFs), i.e., interaction energy patterns, graphically displayed
either as histograms or as 3D color maps, reporting the key
residues involved in the binding with the considered ligands
along with a quantitative estimate of the occurring interactions.
Such analysis provides some information at a glance: On one side,
the identification of the key residues involved in the interactions
highlights possible trends in the binding modes of the considered
derivatives. On the other side, the semiquantitative analysis of
the interactions allows a direct comparison of different ligands
with respect to the quality of ligand−receptor contacts. Figure 1
displays the calculated electrostatic and hydrophobic contribu-
tions to the interaction energy to the hA3 AR for the energetically
more favorable pose of each compound investigated. Most of
the selected poses share a common interacting network with
residues located in TM2, TM3, TM6, TM7, and EL2. The map
of the electrostatic IEFs (Figure 1A) shows an intense band with
negative potential energy (colored in blue) corresponding to
Asn250 (6.55) in TM6, suggesting that this residue is responsible
for the main electrostatic interactions with all the tested
Docking of 5-Alkylamino Derivatives (compounds 5−62).
A first glance at the docking poses of the new derivatives has
revealed that the compounds share at least two common
hypothetical binding modes (Figure 2A,B), which differ in the
orientation of the ligand in the binding cleft: In the first binding
mode (hereafter indicated as BM1), the substituent at the N5
position points away from the binding pocket (Figure 2A),
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the direction of the substituent at the N5 position strongly
depends on its nature and size: Compounds bearing small alkyl
and cycloalkyl groups and polar chains prefer BM1, whereas
the majority of the ligands bearing aromatic rings, bulky
substituents as well as those with the double substitution at the
N5 atom, preferentially adopt BM2. To understand the reasons
for the differences in the direction of the substituents that at the
hA3 AR reflect the above-mentioned binding modes, two
reference compounds (one for each binding mode) have been
selected, and their energetically more favorable docking poses
along with the main interactions occurring between the ligands
and the receptor are discussed herein. Figure 3A,B displays the
hypothetical binding modes of 50 (Ki = 38.6 nM, Figure 2A)
and 35 (Ki = 0.83 nM, Figure 2B) at the hA3 AR as examples of
compounds adopting BM1 and BM2, respectively. In both
cases, the ligand recognition occurs in the upper region of the
TM bundle with the PTP scaffold surrounded by TM3, TM5,
TM6, and TM7, the 2-furyl ring oriented toward TM5. The key
interactions are represented by one and two hydrogen bonds
with Asn250 (6.55) for 50 and 35, respectively, a π−π stacking
interaction between the PTP core and Phe168 (EL2), and
hydrophobic contacts with, among others, Trp243 (6.48) and
Leu246 (6.51). The placement of compound 35 into the binding
cleft resembles that observed for the 1,2,4-triazine adenosine
A2A AR antagonists, that have been recently cocrystallized with
the receptor.35
As stated above, the two BMs differ in the orientation of the
substituent at the N5 position. Figure 2C shows the hA3 AR
embedded in a lipid bilayer simulating the cellular membrane
and helps in visualizing the difference: in BM1 (green arrow)
the substituent at the N5 position is directed away from the
binding cleft, thus being exposed to solvent, whereas in BM2
(orange arrow) the substituent at the N5 position points toward
the binding cavity. As highlighted by the different colors of the
residue side chains in Figure 2C and reported in the IEF analysis
in Figure 2D, the two BMs result from the interaction of the
substituent at the N5 position with different TMs. In particular,
compound 50 (BM1) establishes hydrophobic contacts mainly
with residues in TM6, TM7, and EL2, the most prominent
being Val169 (EL2), Trp243 (6.48), Leu246 (6.51), and Ile268
(7.39). Compound 35, instead, interacts mainly with residues
sited in TM2, TM3, and EL2, such as Leu68 (2.60), Val72
(2.64), Leu90 (3.31), Trp243 (6.48), Leu246 (6.51), and Ile268
(7.39).
The values of the interaction energies in Figure 2D also
reveal that the adoption of BM2 (orange histograms) causes the
ligand to establish a weaker electrostatic interaction with Asn250
(6.55) with respect to BM1 (green histograms), which however
seems to be counter-balanced by stronger hydrophobic contacts
with residues in TM2 and TM3. Of course, this hypothesis
might be expected to be the result of a docking artifact,
considering the well-documented limitations/approximations of
docking methods. Very recently, we presented a novel approach
obtained by the integration of molecular docking and membrane
molecular dynamics simulations with the aim to merge the main
advantage of docking, that is the rapid sampling of ligand poses
into the binding site, with the thermodynamic accuracy of MD
simulations in particular regarding the description, at the
molecular level, of the stability of a GPCR−ligand complex
embedded into an explicit lipid−water environment.36 In this
approach we can try to overcome some of the most crucial
approximations of the conventional scoring functions such as
the absence of explicit water molecules and the explorations of
Figure 2. Schematic representation of the two binding modes BM1
(A) and BM2 (B) inside the hA3 AR. The side chain of Asn250 (6.55)
and Phe168 (EL2) are displayed in ball and stick. (C) Statistical
analysis of the occurrence of the two binding modes for the ligands
showing Ki < 100 nM at both hA3 AR and hA2A AR. The histograms
are color-coded according to the nature of the substituent at the N5
position.
whereas in the other binding mode (hereafter indicated as
BM2) the substituent lies inside the binding cavity (Figure 2B).
The statistical analysis of the occurrence of the two binding
modes (Figure 2C) performed for the compounds showing
Ki < 100 nM reveals an interesting trend: None of the ligands
exhibits BM2 at the hA2A AR, whereas at the hA3 AR, the latter
is the preferred binding mode for the 36% of the compounds.
This suggests that, while the adoption of BM2 might hamper
the ligand to establish interactions with key residues at the hA2A
AR, it might represent an effective binding mode at the hA3 AR.
The color code of the histograms in Figure 2C highlights that
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Figure 3. Hypothetical binding modes of compound 50 (A) and 35 (B) inside the hA3 AR binding site. Poses are viewed from the membrane side
facing TM6, TM7, and TM1 with the view of TM7 partially omitted to aid visualization. The side chains of Asn250 (6.55) and Phe168 (EL2) and
the N5 atom are represented in ball and stick. Hydrogen bond interactions are highlighted as dashed lines, whereas the side chains of residues
interacting with the ligand through hydrophobic contacts are displayed as surfaces. An arrow indicating the directionality of the substituent at the N5
position is shown. (C) Schematic representation of the hA3 AR embedded in a solvated lipid bilayer simulating the cell membrane. The side chains of
residues interacting through hydrophobic contacts are displayed as green and orange ball and sticks, for BM1 and BM2, respectively. Side chains of
Asn250 (6.55), Phe168 (EL2), and Trp243 (6.48) are represented in gray ball and stick. Hydrogen atoms are omitted. (D) IEF analysis for
compounds 50 and 35.
receptor−ligand complex flexibility that have been recently
reported to be very important in determining the binding
energetics of the GPCR−ligand complex.37 Therefore, to
evaluate the reliability of the above-described BMs, we performed
molecular dynamics (MD) simulations and investigated the
evolution of the interactions suggested by the IEF analysis during
a time lapse of 60 ns.
Molecular Dynamics. The ligand−receptor complexes of
the two reference compounds 35 and 50 have been embedded
in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
lipid bilayer and subjected to MD simulations. The analysis of
the MD trajectory and the corresponding IEFs of 50 (Figure S2A,
Supporting Information) highlights that the interaction pattern
that emerged from the analysis of the docking poses is conserved
throughout the simulation. In particular, the interactions with
Asn250 (6.55), Phe168 (EL2), Leu246 (6.51), and Leu90 (3.31)
are constant during the considered time lapse. Moreover, during
the simulation, the PTP core establishes strong hydrogen bond
interactions with Gln167 (data not shown), which is recruited
from EL2 and contributes to further stabilize the ligand−receptor
interaction. These results along with the low average fluctuation
of the ligand (2.28 0.77 Å) supports the high degree of fitness
of the selected starting conformation. The same can be asserted
for 35 (Figure S2B), which shows a strong and persistent
ligand−receptor interaction network with negligible ligand
fluctuations (average 2.21 0.59 Å). Therefore, the favorable
H
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and hydrophobic contacts with the binding pocket delimited by
TM2 and TM3 may be considered a valid rationalization of the
activity of 35 at the hA3 AR. On the whole, the above-described
results have helped in defining the structure affinity profiles of
the new 5-alkylamino scaffold.
into account the different electrostatic potentials of the binding
site entrance and by tailoring the physicochemical properties of
the PTP scaffold accordingly to the desired target.
EXPERIMENTAL SECTION
■
The analysis of the docking poses performed through visual
inspection, statistical analysis of the occurrence of the observed
BMs, and IEF analyses have highlighted that the placement of
the ligands into the binding cavity is mainly driven by the nature
and size of the substituents at the N5 position. In particular,
compounds bearing small alkyl and cycloalkyl groups as well as
polar chains preferentially direct the substituent away from the
binding pocket by adopting BM1, whereas the inclusion of
aromatic rings, bulky substituents, and double substitution at
the N5 atom (also with the inclusion of the nitrogen atom into
certain heterocycles) causes the ligand to adopt a different
placement with the substituent pointing toward regions of the
binding site rich with residues bearing hydrophobic side chains
(BM2). The latter orientation, at the hA3 AR, allows the PTP
to interact with the two key residues Asn250 (6.55) and
Phe168 (EL2) and establish strong and favored interactions
with residues in TM2 and TM3. Conversely, this interaction
pattern has not been observed at the hA2A AR, as all the active
compounds exhibit BM1.
This is consistent with the putative different electrostatic
potentials of the residues that surround the binding cavity in
the two receptor subtypes: In fact, the hA2A AR bears a charged
gate (represented by the ionic couple Glu169 (EL2)-His264
(EL3)) at the binding site entrance that does not allow ligands
with bulky and hydrophobic substituents to approach key
residues in the binding cleft. The hA3 AR binding site, instead,
lacks this ionic gate, as Glu169 is mutated to Val169: Therefore,
according to our proposed model and as emerged from MD
simulations, its binding pocket can accommodate bulky
substituents better than the other AR subtypes. These results
therefore enforces our hypothesis that the presence of the
charged gate at the binding site entrance in hA2A AR might affect
the ligand orientation and its placement into the binding cleft.31
A. Chemistry. Reactions were routinely monitored by thin-layer
chromatography (TLC) on silica gel (precoated F254 Merck plates).
Infrared spectra (IR) were measured on a Jasco FT-IT instrument.
1H NMR were determined in CDCl3 or DMSO-d6 solutions with a
Varian Gemini 200 spectrometer, peaks positions are given in parts per
million (δ) downfield from tetramethylsilane as internal standard, and
J values are given in hertz. Electrospray mass spectra were recorded on
a ESI Bruker 4000 Esquire spectrometer (University of Trieste), and
compounds were dissolved in methanol. Light petroleum ether refers
to the fractions boiling at 40−60 °C. Melting points were determined
on a Buchi-Tottoli instrument and are uncorrected. Flash chromatog-
̈
raphy was performed using Merck 60−200 mesh silica gel. Elemental
analyses were performed by the microanalytical laboratory of
Dipartimento di Chimica, University of Trieste, and were within
0.4% of the theoretical values for C, H, and N.
2-Furan-2-yl-8-methyl-6,8-dihydropyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-one (64). Compound 63 (200 mg, 0.869 mmol)
was dissolved in dry dioxane, and diphosgene was added (1.13 mmol,
0.136 mL). The mixture was heated at 70 °C for 1.5 h. The reaction
was monitored by TLC (EtOAc:MeOH, 9:1), and when the starting
material disappeared, the solvent was removed. Product 64 was
precipitated from EtOAc and petroleum ether, obtaining an ivory
powder that was used without further purification. Yield 46%; ivory solid
1
(EtOAc/petroleum ether) mp >300 °C; H NMR (DMSO-d6) δ: 3.96
(3H, s), 6.71 (1H, dd, J = 2, J = 4), 7.18 (1H, d, J = 4), 7.93 (1H, d, J =
2), 8.58 (1H, s). IR (Nujol) cm−1: 3345−3270, 1695, 1620, 1510, 1430.
ES-MS (methanol): 279.00 (M + 23). Anal. (C11H8N6O2) C, H, N.
5-Chloro-2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidine (65). Compound 64 (100 mg, 0.390 mmol)
was dissolved in phosphoryl chloride (21.46 mmol, 2 mL), and phos-
phorus pentachloride (0.117 mmol, 24 mg) and pyridine (0.975 mmol,
0.079 mL) were added. The mixture was refluxed for 24 h, the solvent
was removed under reduced pressure, and the residue was dissolved in
chloroform and washed with water and brine (1:1). The organic layer
was dried and concentrated, and the residue purified through flash
chromatography using EtOAc:MeOH 9.5:0.5 to afford the desired
compound 65. Yield 34%; white solid (EtOAc/petroleum ether) mp
1
285 °C; H NMR (DMSO-d6) δ: 4.12 (3H, s), 6.73 (1H, dd, J = 2,
CONCLUSIONS
■
J = 4), 7.22 (1H, d, J = 4), 7. 95 (1H, d, J = 2), 8.61 (1H, s). IR
(Nujol) cm−1: 1625, 1500, 1415. ES-MS (methanol): 297.00 (M + 23).
Anal. (C11H7N6OCl) C, H, N.
We have presented a novel series of 5-alkylaminopyrazolo-
[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines acting as antagonists of
the hA3 AR. We have analyzed the corresponding SAR with the
aim to establish the directionality of 5-substitutions inside the
orthosteric binding site of the hA3 AR. Interestingly, almost all
the synthesized compounds showed affinity for the hA3 AR
from nanomolar to subnanomolar range with the most potent
and selective antagonist bearing an (S) α-phenylethylamino
moiety at the 5 position (26, hA3 AR Ki = 0.3 nM). Using an in
silico receptor-driven approach, we have determined the most
favorable orientation of the substitutions at the 5-position of
the PTP scaffold, opening the possibility for further scaffold
decorations that point the N5 position toward the extracellular
environment. In particular, it can be concluded that small alkyl
and cycloalkyl groups as well as polar chains might be suitable
functionalizing moieties at the N5 site for the linkage of
fluorophore groups as well as other “sensoring” moieties that
can be useful for the investigation of the physiological and
physiopathological functions of ARs. Especially, chain-elongated
alkyl groups, as long as not bulky and sterically hindered, might
represent an effective choice. Moreover, in the light of the
obtained results, a higher degree of selectivity toward either the
hA3 AR or the hA2A AR subtype might be afforded by taking
General Procedure for the Synthesis of the 5-Alkoxy-2-furan-2-yl-
8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (3, 4).
To 5 mL of ethanol or methanol was added 8.3 mg (0.364 mmol)
of sodium. When all the sodium was consumed, 100 mg of 5-chloro
derivative 65 was added to the solution (0.364 mmol). Reaction
was refluxed 2 h and monitored through TLC (EtOAc). Then, the
solvent was removed under pressure and the residue purified by flash
chromatography (EtOAc:petroleum ether, 9:1), affording the desired
compounds.
2-(Furan-2-yl)-5-methoxy-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidine (3). Yield 75%; pale brown solid (EtOAc/
petroleum ether) mp 250 °C; 1H NMR (CDCl3) δ: 4.17 (3H, s), 4.36
(3H, s), 6.58 (1H, dd, J = 2, J = 4), 7.26 (1H, d, J = 4), 7.62 (1H, d,
J = 2), 8.18 (1H, s). IR (Nujol) cm−1: 1630, 1510, 1420. Anal.
(C12H10N6O2) C, H, N.
5-Ethoxy-2-(furan-2-yl)-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidine (4). Yield 73%; pale yellow solid (EtOAc/
petroleum ether) mp 223 °C; 1H NMR (CDCl3) δ: 1.43 (3H, t, J = 7),
4.16 (3H, s), 4.40 (2H, q, J = 7), 6.59 (1H, dd, J = 2, J = 4), 7.24 (1H,
d, J = 4), 7. 62 (1H, d, J = 2), 8.16 (1H, s). IR (Nujol) cm−1: 1635,
1515, 1420. Anal. (C13H12N6O2) C, H, N.
General Procedure for the Synthesis of the 5-Amino-Substituted
2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidine (5−62). 5-Chloro derivative 65 (100 mg, 0.364 mmol) was
I
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Journal of Medicinal Chemistry
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1
dissolved in 5 mL of ethanol, triethylamine (TEA, 0.364 mmol, 51 μL)
and the appropriate amine (1.092 mmol) were added, and the mixture
was heated at 110 °C in a sealed tube for 2 h. The reaction was
monitored through TLC (EtOAc:MeOH, 9:1). Then, the solvent was
removed under reduced pressure and the residue purified by flash
chromatography (EtOAc:MeOH 9.5:0.5) affording the desired
compound.
(EtOAc/petroleum ether) mp 114 °C; H NMR (CDCl3) δ: 0.89−
0.98 (6H, m), 1.27−1.53 (8H, m), 1.68−1.81 (1H, m), 3.62 (2H, t,
J = 6), 4.10 (3H, s), 6.22 (1H, bs), 6.58 (1H, dd, J = 2, J = 4), 7.23
(1H, d, J = 4), 7.63 (1H, d, J = 2), 8.10 (1H, s). IR (Nujol) cm−1:
3335−3150, 1645, 1520, 1415. Anal. (C19H25N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-(1,1,3,3-tetramethylbutyl)amine (15). Yield 81%;
pale yellow solid (EtOAc/petroleum ether) mp 176 °C; H NMR
(CDCl3) δ: 1.03 (9H, s), 1.68 (6H, s), 2.04 (2H, s), 4.10 (3H, s), 6.29
(1H, bs), 6.58 (1H, dd, J = 2, J = 4), 7.22 (1H, d, J = 4), 7.62 (1H, d,
J = 2), 8.10 (1H, s). IR (Nujol) cm−1: 3330−3140, 1635, 1520, 1410.
Anal. (C19H25N7O) C, H, N.
1
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)methylamine (5). Yield 75%; pale yellow solid
1
(EtOAc/petroleum ether) mp 285 °C; H NMR (CDCl3) δ: 3.24
(3H, d, J = 5), 4.11 (3H, s), 6.23 (1H, bs), 6.60 (1H, dd, J = 2, J = 4),
7.18 (1H, d, J = 4), 7.61 (1H, d, J = 2), 8.07 (1H, s). IR (Nujol) cm−1:
3350−3170, 1615, 1520, 1410. Anal. (C12H11N7O) C, H, N.
Cyclopropyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (16). Yield 88%; yellow solid
Ethyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)amine (6). Yield 83%; pale yellow solid
1
(EtOAc/petroleum ether) mp 280 °C; H NMR (CDCl3) δ: 0.75−
1
(EtOAc/petroleum ether) mp 256 °C; H NMR (CDCl3) δ: 1.38
0.82 (2H, m), 0.95−1.00 (2H, m), 2.95−3.15 (1H, m), 4.12 (3H, s),
6.43 (1H, bs), 6.60 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4), 7.62
(1H, d, J = 2), 8.09 (1H, s). IR (Nujol) cm−1: 3340−3160, 1620, 1505,
1425. Anal. (C14H13N7O) C, H, N.
(3H, t, J = 7), 3.68−3.78 (2H, m), 4.10 (3H, s), 6.21 (1H, bs), 6.58 (1H,
dd, J = 2, J = 4), 7.18 (1H, d, J = 4), 7.62 (1H, d, J = 2), 8.07 (1H, s). IR
(Nujol) cm−1: 3330.3180, 1630, 1505, 1420. ES-MS (methanol) m/z:
284.10 (M + 1),306.10 (M + 23). Anal. (C13H13N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)isopropylamine (7). Yield 78%; pale yellow solid
Cyclobutyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (17). Yield 80%; light brown
solid (EtOAc/petroleum ether) mp 250 °C; H NMR (CDCl3) δ:
1
1
(EtOAc/petroleum ether) mp 214 °C; H NMR (CDCl3) δ: 1.40
1.73−1.91 (2H, m), 1.98−2.21 (2H, m), 2.45−2.63 (2H, m), 4.10
(3H, s), 4.71−4.83 (1H, m), 6.37 (1H, d, J = 7), 6.60 (1H, dd, J = 2,
J = 4), 7.21 (1H, d, J = 4), 7.63 (1H, d, J = 2), 8.06 (1H, s). IR (Nujol)
cm−1: 3350−3170, 1620, 1510, 1425. Anal. (C15H15N7O) C, H, N.
Cyclopentyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (18). Yield 76%; pale brown solid
(6H, d, J = 6), 4.11 (3H, s), 4.46−4.47 (1H, m), 6.08 (1H, d, J = 8),
6.60 (1H, dd, J = 2, J = 4), 7.22 (1H, d, J = 4), 7.63 (1H, d, J = 2), 8.10
(1H, s). IR (Nujol) cm−1: 3340−3150, 1625, 1510, 1415. Anal.
(C14H15N7O) C, H, N.
(R,S)-(+,−)-sec-Butyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
1
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (8). Yield 77%; white solid
(EtOAc/petroleum ether) mp 226 °C; H NMR (CDCl3) δ: 1.59−
1
(EtOAc/petroleum ether) mp 190 °C; H NMR (CDCl3) δ: 1.01
2.00 (6H, m), 2.14−2.34 (2H, m), 4.10 (3H, s), 4.54−4.62 (1H, m),
6.18 (1H, d, J = 7), 6.59 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4), 7.62
(1H, d, J = 2), 8.06 (1H, s). IR (Nujol) cm−1: 3345−3160, 1640, 1530,
1410. Anal. (C16H17N7O) C, H, N.
(3H, t, J = 7), 1.35 (3H, t, J = 7), 1.60−1.83 (2H, m), 4.10 (3H, s),
4.25−4.45 (1H, m), 6.05 (1H, d, J = 8), 6.59 (1H, dd, J = 2, J = 4),
7.20 (1H, d, J = 4), 7.62 (1H, d, J = 2), 8.08 (1H, s). IR (Nujol) cm−1:
3335−3165, 1615, 1510, 1405. Anal. (C15H17N7O) C, H, N.
Cyclohexyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (19). Yield 63%; brown solid
(S)-(+)-sec-Butyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (9). Yield 65%; old pink
1
(EtOAc/petroleum ether) mp 188 °C; H NMR (CDCl3) δ: 1.25−
1
solid (EtOAc/petroleum ether) mp 162 °C; H NMR (CDCl3) δ:
1.92 (8H, m), 2.15−2.24 (2H, m), 4.09−4.19 (4H, m), 6.10 (1H, bs),
6.58 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4), 7.62 (1H, d, J = 2),
8.06 (1H, s). IR (Nujol) cm−1: 3340−3150, 1625, 1510, 1415. Anal.
(C17H19N7O) C, H, N.
1.01 (3H, t, J = 7), 1.35 (3H, t, J = 7), 1.59−1.85 (2H, m), 4.11 (3H, s),
4.27−4.45 (1H, m), 6.07 (1H, d, J = 8), 6.60 (1H, dd, J = 2, J = 4), 7.30
(1H, d, J = 4), 7.64 (1H, d, J = 2), 8.19 (1H, s). IR (Nujol) cm−1:
3345−3160, 1625, 1520, 1405. Anal. (C15H17N7O) C, H, N.
Cycloheptyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (20). Yield 76%; orange solid
(R)-(−)-sec-Butyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
1
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (10). Yield 82%; old pink
(EtOAc/petroleum ether) mp 130 °C; H NMR (CDCl3) δ: 1.50−
1
solid (EtOAc/petroleum ether) mp 180 °C; H NMR (CDCl3) δ:
1.89 (10H, m), 2.15−2.31 (2H, m), 4.10 (3H, s), 4.32−4.45 (1H, m),
6.19 (1H, d, J = 8), 6.60 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4), 7.63
(1H, d, J = 2), 8.07 (1H, s). IR (Nujol) cm−1: 3335−3140, 1620, 1530,
1435. Anal. (C18H21N7O) C, H, N.
1.01 (3H, t, J = 7), 1.35 (3H, t, J = 7), 1.59−1.83 (2H, m), 4.10
(3H, s), 4.23−4.45 (1H, m), 6.07 (1H, d, J = 8), 6.60 (1H, dd, J = 2,
J = 4), 7.28 (1H, d, J = 4), 7.64 (1H, d, J = 2), 8.17 (1H, s). IR (Nujol)
cm−1: 3335−3150, 1630, 1500, 1415. Anal. (C15H17N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)pentylamine (11). Yield 74%; pale yellow solid
Cyclooctyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (21). Yield 78%; brown solid
1
(EtOAc/petroleum ether) mp 149 °C; H NMR (CDCl3) δ: 1.61−
1
(EtOAc/petroleum ether) mp 157 °C; H NMR (CDCl3) δ: 1.01
2.15 (14H, m), 4.10 (3H, s), 4.35−4.53 (1H, m), 6.19 (1H, d, J = 8),
6.60 (1H, dd, J = 2, J = 4), 7.21 (1H, d, J = 4), 7.63 (1H, d, J = 2),
8.07 (1H, s). IR (Nujol) cm−1: 3355−3160, 1635, 1505, 1425. Anal.
(C19H23N7O) C, H, N.
(3H, t, J = 7), 1.33−1.52 (4H, m), 1.67−1.83 (2H, m), 3.67 (2H, q,
J = 7), 4.10 (3H, s), 6.26 (1H, bs), 6.59 (1H, dd, J = 2, J = 4), 7.22
(1H, d, J = 4), 7.62 (1H, d, J = 2), 8.09 (1H, s). IR (Nujol) cm−1:
3340−3160, 1615, 1520, 1415. Anal. (C16H19N7O) C, H, N.
Cyclohexylmethyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (22). Yield 74%; brown
solid (EtOAc/petroleum ether) mp 169 °C; H NMR (CDCl3) δ:
0.83−1.39 (4H, m), 1.55−1.93 (6H, m), 2.76 (1H, d, J = 7), 3.53
(2H, t, J = 7), 4.10 (3H, s), 6.33 (1H, bs), 6.60 (1H, dd, J = 2, J = 4),
7.21 (1H, d, J = 4), 7.63 (1H, d, J = 2), 8.07 (1H, s). IR (Nujol) cm−1:
3355−3150, 1625, 1510, 1430. Anal. (C18H21N7O) C, H, N.
Adamantan-1-yl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (23). Yield 75%; pale
yellow solid (EtOAc/petroleum ether) mp 282 °C; H NMR (CDCl3)
δ: 1.67−1.83 (6H, m), 2.17 (3H, s), 2.32 (6H, s), 4.10 (3H, s), 6.14
(1H, bs), 6.60 (1H, dd, J = 4, J = 2), 7.32 (1H, d, J = 4), 7.63 (1H, d,
J = 2), 8.13 (1H, s). IR (Nujol) cm−1: 3345−3170, 1635, 1520, 1405.
Anal. (C21H23N7O) C, H, N.
Adamantan-2-yl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (24). Yield 83%; pale
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-(1-methylhexyl)amine (12). Yield 88%; yellow solid
1
1
(EtOAc/petroleum ether) mp 150 °C; H NMR (CDCl3) δ: 0.87
(3H, t, J = 7), 1.19−1.73 (11H, m), 4.10 (3H, s), 4.33−4.56 (1H, m),
6.04 (1H, d, J = 8), 6.59 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4), 7.62
(1H, d, J = 2), 8.07 (1H, s). IR (Nujol) cm−1: 3350−3150, 1625, 1500,
1420. Anal. (C18H23N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
1
pyrimidin-5-yl)-(1-methylheptyl)amine (13). Yield 84%; yellow solid
1
(EtOAc/petroleum ether) mp 149 °C; H NMR (CDCl3) δ: 0.85
(3H, t, J = 7), 1.13−1.55 (11H, m), 1.57−1.73 (2H, m), 4.09 (3H, s),
4.33−4.55 (1H, m), 5.81 (1H, d, J = 9), 6.60 (1H, dd, J = 2, J = 4),
7.22 (1H, d, J = 4), 7.63 (1H, d, J = 2), 8.09 (1H, s). IR (Nujol) cm−1:
3345−3165, 1615, 1510, 1400. Anal. (C19H25N7O) C, H, N.
(2-Ethylhexyl)(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (14). Yield 72%; pale yellow solid
1
yellow solid (EtOAc/petroleum ether) mp 286 °C; H NMR (CDCl3)
J
dx.doi.org/10.1021/jm300899q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
δ: 1.68−2.04 (12H, m), 2.24 (2H, s), 4.09 (3H, s), 4.44 (1H, bs), 6.53−
6.58 (2H, m), 7.23 (1H, d, J = 4), 7.63 (1H, d, J = 2), 8.09 (1H, s).
IR (Nujol) cm−1: 3330−3140, 1640, 1520, 1405. Anal. (C21H23N7O)
C, H, N.
Benzyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)amine (25). Yield 87%; yellow solid (EtOAc/
petroleum ether) mp 188 °C; 1H NMR (CDCl3) δ: 4.12 (3H, s), 4.87
(2H, d, J = 5), 5.56−5.62 (2H, m), 7.17−7.46 (6H, m), 7.69 (1H, d,
J = 2), 8.09 (1H, s). IR (Nujol) cm−1: 3350−3160, 1630, 1515, 1425.
Anal. (C18H15N7O) C, H, N.
δ: 4.10 (3H, s), 6.61 (1H, dd, J = 2, J = 4), 6.74 (1H, d, J = 8), 6.86
(1H, d, J = 8), 7.31−7.38 (11H, m), 7.64 (1H, d, J = 2), 8.22 (1H, s).
IR (Nujol) cm−1: 3350−3160, 1655, 1540, 1435. Anal. (C24H19N7O)
C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-2-phenethylamine (36). Yield 76%; pale brown solid
1
(EtOAc/petroleum ether) mp 120 °C; H NMR (CDCl3) δ: 3.08
(2H, t, J = 7), 3.89−3.96 (2H, m), 4.11 (3H, s), 5.34 (1H, bs), 6.58
(1H, dd, J = 2, J = 4), 7.16−7.37 (6H, m), 7.59 (1H, d, J = 2), 8.09
(1H, s). IR (Nujol) cm−1: 3330−3155, 1620, 1525, 1405. Anal.
(C19H17N7O) C, H, N.
(S)-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-yl)-(1-phenylethyl)amine (26). Yield 84%; pale yellow
[2-(3,4-Dimethoxyphenyl)ethyl](2-furan-2-yl-8-methyl-8H-
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (37). Yield
89%; pale brown solid (EtOAc/petroleum ether) mp 165 °C;
1H NMR (CDCl3) δ: 3.01 (2H, t, J = 7), 3.87−3.97 (8H, m), 4.12
(3H, s), 6.35 (1H, bs), 6.59 (1H, dd, J = 2, J = 4), 6.81 (3H, m), 7.18
(1H, d, J = 4), 7.61 (1H, d, J = 2), 8.09 (1H, s). IR (Nujol) cm−1:
3340−3165, 1620, 1505, 1420. Anal. (C21H21N7O3) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-(1-methyl-3-phenylpropyl)amine (38). Yield 71%;
1
solid (EtOAc/petroleum ether) mp 189 °C; H NMR (CDCl3) δ:
1.72 (3H, d, J = 7), 4.09 (3H, s), 5.49−5.63 (1H, m), 6.48 (1H, bs),
6.58 (1H, dd, J = 2, J = 4), 7.19−7.39 (4H, m), 7.50 (2H, d, J = 7),
7.61 (1H, d, J = 2), 8.06 (1H, s). IR (Nujol) cm−1: 3350−3170, 1640,
1510, 1425. Anal. (C19H17N7O) C, H, N.
(R)-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-yl)(1-phenylethyl)amine (27). Yield 92%; yellow solid
(EtOAc/petroleum ether) mp 69 °C; 1H NMR (CDCl3) δ: 1.72
(3H, d, J = 7), 4.09 (3H, s), 5.47−5.62 (1H, m), 6.48−6.63 (2H, m),
7.20−7.51 (6H, m), 7.61 (1H, d, J = 2), 8.06 (1H, s). IR (Nujol)
cm−1: 3350−3130, 1635, 1525, 1400. Anal. (C19H17N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)(4-methylbenzyl)amine (28). Yield 79%; pale brown
1
pale yellow solid (EtOAc/petroleum ether) mp 177 °C; H NMR
(CDCl3) δ: 1.40 (3H, d, J = 7), 1.99−2.09 (2H, m), 2.78 (2H, t, J = 8),
4.11 (3H, s), 4.51 (1H, m), 6.08 (1H, d, J = 8), 6.60 (1H, dd, J = 2,
J = 4), 7.14−7.21 (6H, m), 7.63 (1H, d, J = 2), 8.08 (1H, s). IR (Nujol)
cm−1: 3350−3165, 1630, 1520, 1415. Anal. (C21H21N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-[2-(1H-indol-3-yl)ethyl]amine (39). Yield 67%; pale
brown solid (EtOAc/petroleum ether) mp 160 °C; 1H NMR (CDCl3)
δ: 3.25 (2H, t, J = 7), 4.00−4.14 (5H, m), 6.54 (1H, bs), 6.60 (1H, dd,
J = 2, J = 4), 6.93−7.30 (3H, m), 7.39 (2H, d, J = 6), 7.50−7.68
(2H, m), 8.11 (1H, bs), 8.33 (1H, s). IR (Nujol) cm−1: 3370−3130,
1645, 1530, 1460. Anal. (C21H18N8O) C, H, N.
1
solid (EtOAc/petroleum ether) mp 160 °C; H NMR (CDCl3) δ:
2.45 (3H, s), 4.12 (3H, s), 5.82 (2H, d, J = 7), 6.60−6.68 (2H, m),
7.03−7.48 (5H, m), 7.70 (1H, d, J = 2), 8.19 (1H, s). IR (Nujol)
cm−1: 3340−3170, 1645, 1525, 1405. Anal. (C19H17N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)(4-methoxybenzyl)amine (29). Yield 82%; pale orange
1
solid (EtOAc/petroleum ether) mp 175 °C; H NMR (CDCl3) δ:
3.80 (3H, s), 4.12 (3H, s), 4.79 (2H, d, J = 5), 6.50 (1H, bs), 6.58 (1H,
dd, J = 4, J = 2), 6.89 (2H, d, J = 8), 7.23 (1H, d, J = 4), 7.39 (2H, d,
J = 8), 7.61 (1H, d, J = 2), 8.16 (1H, s). IR (Nujol) cm−1: 3345−3150,
1625, 1535, 1415. Anal. (C19H17N7O2) C, H, N.
2-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)ethanol (40). Yield 67%; yellow solid
1
(EtOAc/petroleum ether) mp 188 °C H NMR (CDCl3) δ: 3.88−
4.06 (4H, m), 4.11 (3H, s), 6.62 (1H, dd, J = 2, J = 4), 7.30 (1H, d,
J = 4), 7.65 (1H, d, J = 2), 8.18 (1H, s). ES-MS (methanol): 300.1
(M + 1), 322.1 (M + 23). IR (Nujol) cm−1: 3550−3130, 1620, 1525,
1405. Anal. (C13H13N7O2) C, H, N.
(4-Chlorobenzyl)-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (30). Yield 79%; pale
1
yellow solid (EtOAc/petroleum ether) mp 212 °C; H NMR (CDCl3)
δ: 4.12 (3H, s), 4.84 (2H, d, J = 6), 6.55−6.63 (2H, m), 7.21−7.43 (5H,
m), 7.61 (1H, d, J = 2), 8.12 (1H, s). IR (Nujol) cm−1: 3340−3130,
1625, 1530, 1420. Anal. (C18H14N7OCl) C, H, N.
3-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)propan-1-ol (41). Yield 87%; pale yellow solid
1
(EtOAc/petroleum ether) mp 185 °C; H NMR (CDCl3) δ: 1.97
(2H, t, J = 6), 3.76−3.94 (4H, m), 4.13 (3H, s), 4.50 (1H, bs), 6.53
(1H, dd, J = 2, J = 4), 6.74 (1H, bs), 7.40 (1H, d, J = 4), 7.66 (1H, d,
J = 2), 8.31 (1H, s). IR (Nujol) cm−1: 3400−3130, 1640, 1535, 1415.
Anal. (C14H15N7O2) C, H, N.
(4-Fluorobenzyl)(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (31). Yield 84%; pale
1
yellow solid (EtOAc/petroleum ether) mp 186 °C; H NMR (CDCl3)
δ: 4.12 (3H, s), 4.94 (2H, d, J = 6), 6.57−6.59 (2H, m), 6.99−7.08
(2H, m), 7.22 (1H, d, J = 4), 7.41−7.47 (2H, m), 7.61 (1H, d, J = 2),
8.13 (1H, s). IR (Nujol) cm−1: 3345−3150, 1630, 1515, 1425. Anal.
(C18H14N7OF) C, H, N.
1-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)propan-2-ol (42). Yield 73%; brown solid
1
(EtOAc/petroleum ether) mp 215 °C; H NMR (CDCl3) δ: 1.32
(3H, d, J = 6), 3.44−3.61 (1H, m), 3.83−3.99 (1H, m), 4.10 (3H, s),
4.11−4.31 (1H, m), 6.60−6.64 (2H, m), 7.21 (1H, d, J = 4), 7.62
(1H, d, J = 2), 8.07 (1H, s). IR (Nujol) cm−1: 3450−3110, 1650, 1505,
1430. ES-MS (methanol) m/z: 336.1 (M + 23). Anal. (C14H15N7O2)
C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)(4-trifluoromethylbenzyl)amine (32). Yield 78%; pale
yellow solid (EtOAc/petroleum ether) mp 172 °C; 1H NMR (CDCl3)
δ: 4.12 (3H, s), 4.94 (2H, d, J = 6), 6.58 (1H, dd, J = 2, J = 4), 6.67
(1H, bs), 7.21 (1H, d, J = 4), 7.59 (5H, m), 8.12 (1H, s). IR (Nujol)
cm−1: 3350−3170, 1650, 1535, 1415. Anal. (C19H14N7OF3) C, H, N.
(3,4-Dimethoxybenzyl)(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (33). Yield 74%; yellow
2-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)butan-1-ol (43). Yield 75%; yellow solid
1
(EtOAc/petroleum ether) mp 161 °C; H NMR (CDCl3) δ: 1.07
1
solid (EtOAc/petroleum ether) mp 109 °C; H NMR (CDCl3) δ:
(3H, t, J = 7), 1.83 (2H, m), 3.80−3.85 (1H, m), 3.96−4.08 (1H, m),
4.13 (3H, s), 4.38−4.74 (2H, m), 6.45 (1H, bs), 6.63 (1H, dd, J = 2,
J = 4), 7.39 (1H, d, J = 4), 7.67 (1H, d, J = 2), 8.27 (1H, s). IR (Nujol)
cm−1: 3400−3130, 1625, 1540, 1430. Anal. (C15H17N7O2) C, H, N.
2-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)propane-1,3-diol (44). Yield 61%; yellow
3.88 (6H, s), 4.12 (3H, s), 4.80 (2H, d, J = 5), 6.45−6.62 (2H, m),
6.83 (1H, d, J = 7), 6.93−7.05 (2H, m), 7.19 (1H, d, J = 4), 7.60 (1H,
d, J = 2), 8.10 (1H, s). IR (Nujol) cm−1: 3340−3145, 1625, 1525,
1415. Anal. (C20H19N7O3) C, H, N.
Biphenyl-4-ylmethyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e]-
1
[1,2,4]triazolo[1,5-c]pyrimidin-5-yl)amine (34). Yield 82%; yellow
solid (EtOAc/petroleum ether) mp 215 °C; H NMR (CDCl3) δ:
1
solid (EtOAc/petroleum ether) mp 240 °C; H NMR (CDCl3) δ:
3.66 (4H, bs), 4.02 (3H, s), 4.17 (1H, m), 4.90 (2H, bs), 6.73 (1H, dd,
J = 2, J = 4), 7.07 (1H, d, J = 8), 7.23 (1H, d, J = 4), 7.94 (1H, d, J =
2), 8.59 (1H, s). IR (Nujol) cm−1: 3550−3060, 1625, 1505, 1435.
Anal. (C14H15N7O3) C, H, N.
4.13 (3H, s), 4.92 (2H, d, J = 6), 6.57−6.59 (2H, m), 7.19−7.60 (11H,
m), 8.11 (1H, s). IR (Nujol) cm−1: 3355−3180, 1650, 1535, 1405.
Anal. (C24H19N7O) C, H, N.
Benzhydryl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (35). Yield 64%; pale yellow
solid (EtOAc/petroleum ether) mp 205 °C; 1H NMR (CDCl3)
4-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)butan-1-ol (45). Yield 78%; yellow
solid (EtOAc/petroleum ether) mp 161 °C; 1H NMR (CDCl3)
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dx.doi.org/10.1021/jm300899q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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1
δ: 1.74−1.99 (4H, m), 3.76 (4H, bs), 4.12 (3H, s), 6.59−6.62 (2H,
m), 7.34 (1H, d, J = 4), 7.65 (1H, d, J = 2), 8.24 (1H, s). IR (Nujol)
cm−1: 3400−3120, 1635, 1520, 1425. Anal. (C15H17N7O2) C, H, N.
5-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)pentan-1-ol (46). Yield 67%; pale brown solid
(EtOAc/petroleum ether) mp 81 °C; 1H NMR (CDCl3) δ 1.48−1.84
(6H, m), 3.65−3.75 (4H, m), 4.10 (3H, s), 6.34 (1H, bs), 6.59 (1H,
dd, J = 2, J = 4), 7.23 (1H, d, J = 4), 7.62 (1H, d, J = 2), 8.11 (1H, s).
IR (Nujol) cm−1: 3350−3080, 1615, 1530, 1420. Anal. (C16H19N7O2)
C, H, N.
(EtOAc/petroleum ether) mp 145 °C; H NMR (CDCl3) δ: 3.34
(3H, s), 4.13 (3H, s), 5.22 (2H, s), 6.56 (1H, dd, J = 2, J = 4), 7.16
(1H, d, J = 4), 7.30−7.40 (5H, m), 7.59 (1H, d, J = 2), 8.17 (1H, s). IR
(Nujol) cm−1: 1650, 1515, 1430. Anal. (C19H17N7O) C, H, N.
Dibenzyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (57). Yield 62%; white solid
1
(EtOAc/petroleum ether) mp 108 °C; H NMR (CDCl3) δ: 4.13
(3H, s), 5.15 (4H, s), 6.54 (1H, dd, J = 2, J = 4), 7.00 (1H, d, J = 4),
7.31−7.35 (10H, m), 7.59 (1H, d, J = 2), 8.17 (1H, s). IR (Nujol)
cm−1: 1655, 1525, 1430. Anal. (C25H21N7O) C, H, N.
2-[Ethyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)amino]ethanol (58). Yield 88%; pale yellow
6-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidin-5-ylamino)hexan-1-ol (47). Yield 82%; orange solid
1
1
(EtOAc/petroleum ether) mp 139 °C; H NMR (CDCl3) δ: 1.48−
solid (EtOAc/petroleum ether) mp 183 °C; H NMR (CDCl3) δ:
1.79 (8H, m), 3.63−3.75 (4H, m), 4.11 (3H, s), 6.30 (1H, bs), 6.61
(1H, dd, J = 2, J = 4), 7.30 (1H, d, J = 4), 7.64 (1H, d, J = 2), 8.14
(1H, s). IR (Nujol) cm−1: 3390−3100, 1650, 1525, 1415. Anal.
(C17H21N7O2) C, H, N.
1.32 (3H, t, J = 7), 3.53−3.62 (4H, m), 3.88 (3H, s), 4.62 (2H, t,
J = 8), 6.51 (1H, dd, J = 2, J = 4), 7.03 (1H, d, J = 4), 7.53 (1H, d,
J = 2), 8.00 (1H, s). IR (Nujol) cm−1: 3450−3110, 1625, 1535, 1425.
Anal. (C15H17N7O2) C, H, N.
N-(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-
2-Furan-2-yl-8-methyl-5-pyrrolidin-1-yl-8H-pyrazolo[4,3-e]-
c]pyrimidin-5-yl)-O-methylhydroxylamine (48). Yield 54%; yellow
[1,2,4]triazolo[1,5-c]pyrimidine (59). Yield 91%; pale yellow solid
1
solid (EtOAc/petroleum ether) mp 205 °C; H NMR (CDCl3) δ:
1
(EtOAc/petroleum ether) mp 270 °C; H NMR (CDCl3) δ: 2.03
4.01−4.04 (6H, m), 6.57 (1H, dd, J = 2, J = 4), 7.20 (1H, d, J = 4),
7.60 (1H, d, J = 2), 7.90 (1H, s), 8.40 (1H, bs). IR (Nujol) cm−1:
3350−3150, 1630, 1515, 1405. Anal. (C12H11N7O) C, H, N.
(4H, bs), 4.03−4.12 (7H, m), 6.57 (1H, dd, J = 2, J = 4), 7.18 (1H, d,
J = 4), 7.61 (1H, d, J = 2), 8.06 (1H, s). IR (Nujol) cm−1: 1630, 1515,
1420. Anal. (C15H15N7O) C, H, N.
2-Furan-2-yl-8-methyl-5-piperidin-1-yl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidine (60). Yield 89%; pale yellow solid (EtOAc/
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-(2-methoxyethyl)amine (49). Yield 72%; brown solid
1
(EtOAc/petroleum ether) mp 105 °C; H NMR (CDCl3) δ: 3.32
1
petroleum ether) mp 181 °C; H NMR (CDCl3) δ: 1.76 (6H, bs),
(3H, s), 3.69−4.00 (4H, m), 4.11 (3H, s), 6.60 (2H, m), 7.24 (1H, d,
J = 4), 7.63 (1H, d, J = 2), 8.11 (1H, s). IR (Nujol) cm−1: 3400−3160,
1635, 1505, 1420. Anal. (C14H15N7O2) C, H, N.
3.92−3.95 (4H, m), 4.13 (3H, s), 6.58 (1H, dd, J = 2, J = 4), 7.22
(1H, d, J = 4), 7.62 (1H, d, J = 2), 8.14 (1H, s). IR (Nujol) cm−1:
1640, 1525, 1405. Anal. (C16H17N7O) C, H, N.
2-Furan-2-yl-8-methyl-5-(4-methyl-piperazin-1-yl)-8H-pyrazolo-
[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (61). Yield 76%; pale yellow
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
pyrimidin-5-yl)-(2-phenoxyethyl)amine (50). Yield 54%; brown solid
1
(EtOAc/petroleum ether) mp 140 °C; H NMR (CDCl3) δ: 4.05−
1
solid (EtOAc/petroleum ether) mp 221 °C; H NMR (CDCl3) δ:
4.17 (5H, m), 4.24−4.32 (2H, m), 6.59 (1H, dd, J = 2, J = 4), 6.70
(1H, bs), 6.91−6.98 (3H, m), 7.23−7.24 (3H, m), 7.63 (1H, d, J = 2),
8.12 (1H, s). IR (Nujol) cm−1: 3340−3120, 1650, 1545, 1435. Anal.
(C19H17N7O2) C, H, N.
2.56 (3H, s), 2.81−3.03 (4H, m), 4.15−4.33 (7H, m), 6.58 (1H, dd,
J = 2, J = 4), 7.19 (1H, d, J = 4), 7.62 (1H, d, J = 2), 8.14 (1H, s). IR
(Nujol) cm−1: 1625, 1530, 1415. Anal. (C16H18N8O) C, H, N.
2-Furan-2-yl-8-methyl-5-morpholin-4-yl-8H-pyrazolo[4,3-e]-
[1,2,4]triazolo[1,5-c]pyrimidine (62). Yield 93%; pale orange solid
Dimethyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)amine (51). Yield 65%; yellow solid
1
1
(EtOAc/petroleum ether) mp 246 °C; H NMR (CDCl3) δ: 3.94−
(EtOAc/petroleum ether) mp 171 °C; H NMR (CDCl3) δ: 3.45
4.05 (8H, m), 4.14 (3H, s), 6.58 (1H, dd, J = 2, J = 4), 7.22 (1H, d,
J = 4), 7.63 (1H, d, J = 2), 8.17 (1H, s). IR (Nujol) cm−1: 1630, 1525,
1430. Anal. (C15H15N7O2) C, H, N.
(6H, s), 4.13 (3H, s), 6.59 (1H, dd, J = 2, J = 4), 7.24 (1H, d, J = 4),
7.63 (1H, d, J = 2), 8.22 (1H, s). IR (Nujol) cm−1: 1630, 1515, 1425.
Anal. (C13H13N7O) C, H, N.
B. Biological Activity. Binding at Human A1, A2A, and A3
Adenosine Receptors. All pharmacological methods followed the
procedures as described earlier.30 In brief, membranes for radioligand
binding were prepared from CHO cells stably transfected with human
adenosine receptor subtypes in a two-step procedure. In a first low-
speed step (1000g) cell fragments and nuclei were removed. The crude
membrane fraction was sedimented from the supernatant at 100 000g.
The membrane pellet was resuspended in the buffer used for the
respective binding experiments, frozen in liquid nitrogen, and stored at
80 °C. For the measurement of adenylyl cyclase activity, only one high
speed centrifugation of the homogenate was used. The resulting crude
membrane pellet was resuspended in 50 mM Tris/HCl, pH 7.4, and
immediately used for the cyclase assay.
Butyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)methylamine (52). Yield 76%; pale yellow solid
1
(EtOAc/petroleum ether) mp 151 °C; H NMR (CDCl3) δ: 0.97
(3H, t, J = 7), 1.36−1.46 (2H, m), 1.76−1.83 (2H, m), 3.44 (3H, s),
3.94 (2H, t, J = 7), 4.14 (3H, s), 6.63 (1H, dd, J = 2, J = 4), 7.49 (1H,
d, J = 4), 7.67 (1H, d, J = 2), 8.48 (1H, s). IR (Nujol) cm−1: 1625,
1535, 1415. Anal. (C16H19N7O) C, H, N.
Diethyl-(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)amine (53). Yield 95%; yellow solid (EtOAc/
petroleum ether) mp 165 °C; 1H NMR (CDCl3) δ: 1.38 (6H, t, J = 7),
3.95 (4H, q, J = 7), 4.10 (3H, s), 6.58 (1H, dd, J = 2, J = 4), 7.19 (1H,
d, J = 4), 7.61 (1H, d, J = 2), 8.12 (1H, s). IR (Nujol) cm−1: 1620,
1530, 1405. Anal. (C15H17N7O) C, H, N.
(2-Furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]-
For radioligand binding at A1 adenosine receptors, 1 nM [3H]CCPA
was used, whereas 30 and 10 nM [3H]NECA were used for A2A and
A3 receptors, respectively. Nonspecific binding of [3H]CCPA was
determined in the presence of 1 mM theophylline, and in the case of
[3H]NECA 100 μM R-PIA was used.38
pyrimidin-5-yl)dipentylamine (54). Yield 81%; pale yellow solid
1
(EtOAc/petroleum ether) mp 106 °C; H NMR (CDCl3) δ: 0.91
(6H, t, J = 7), 1.34−1.46 (8H, m), 1.68−1.87 (4H, m), 3.87 (4H, q,
J = 7), 4.10 (3H, s), 6.59 (1H, dd, J = 2, J = 4), 7.18 (1H, d, J = 4),
7.62 (1H, d, J = 2), 8.12 (1H, s). IR (Nujol) cm−1: 1625, 1515, 1415.
Anal. (C21H29N7O) C, H, N.
Adenylyl Cyclase Activity. The potency of antagonists at the A2B
receptor was determined in adenylyl cyclase experiments. The
procedure was carried out as described previously with minor
modifications.30,39 Membranes were incubated with about 150 000
cpm of [α-32P]ATP for 20 min in the incubation mixture as described
without EDTA and NaCl.39,40 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 was calculated accordingly. Dissociation
Cyclohexyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]-
triazolo[1,5-c]pyrimidin-5-yl)methylamine (55). Yield 78%; pale
yellow solid (EtOAc/petroleum ether) mp 178 °C; 1H NMR
(CDCl3) δ: 1.42−1.74 (4H, m), 1.85−2.04 (6H, m), 3.28 (3H, s),
4.11 (3H, s), 4.71 (1H, t, J = 7), 6.58 (1H, dd, J = 2, J = 4), 7.19
(1H, d, J = 4), 7.62 (1H, d, J = 2), 8.12 (1H, s). IR (Nujol) cm−1:
1630, 1535, 1425. Anal. (C18H21N7O) C, H, N.
Benzyl(2-furan-2-yl-8-methyl-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)methylamine (56). Yield 78%; pale yellow solid
L
dx.doi.org/10.1021/jm300899q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
constants (Ki) for antagonists were then calculated with the Cheng
and Prusoff equation.40
and the pressure was maintained at 1 atm using a Berendensen
barostat. Bond lengths involving hydrogen atoms were constrained
using the M-SHAKE56 algorithm with an integration time step of 2 fs.
Harmonical constraints were then removed during the 60 ns of
the NVT ensemble. Long-range Coulomb interactions were handled
using the particle mesh Ewald summation method (PME)57 with grid
size rounded to the approximate integer value of cell wall dimensions.
A nonbonded cutoff distance of 9 Å with a switching distance of 7.5 Å
was used.
C. Molecular Modeling. Modeling studies were carried out on a
20 CPU (Intel Core2 Quad CPU 2.40 GHz) Linux cluster. Energy
calculations and analyses of docking poses were performed with the
Molecular Operating Environment (MOE, version 2010.10) suite.41
Quantum mechanical calculations were carried out with the software
package MOPAC (version 7), as implemented in MOE.42 Gold suite
(version 5.0) was used to perform all the docking simulations.43
Three Dimensional Structures of hA2A AR, and hA3 AR. The
crystallographic structure of hA2A AR, in complex with the high affinity
antagonist ZM241385 (PDB access code: 3EML),32 and a previously
reported homology model of the hA3 AR31 were used to perform
molecular docking studies at these receptor subtypes. The numbering
of the amino acids follows the arbitrary scheme by Ballesteros and
Weinstein: each amino acid identifier starts with the helix number,
followed by the position relative to a reference residue among the
most conserved amino acids in that helix, to which the number 50 is
arbitrarily assigned.44
ASSOCIATED CONTENT
■
S
* Supporting Information
Elemental analyses and additional modeling figures and videos.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Molecular Docking of hA3 AR Antagonists. Ligand structures were
built using the MOE-builder tool, as part of the MOE suite,41 and were
subjected to a MMFF94x energy minimization until the rms conjugate
gradient was <0.05 kcal mol−1 Å−1. The validation of the proposed
docking protocol has been previously published.45
*Tel +39 040 5583726 (G.S.), +39 049 8275704 (S.M.). Fax
+39 040 52572 (G.S.), +39 049 8275366 (S.M.). E-mail:
spalluto@units.it (G.S.); stefano.moro@unipd.it (S.M.).
Notes
All antagonists were docked into the hypothetical TM binding site
of the hA3 AR model and the orthosteric binding site of the hA2A AR
crystal structure, by employing the docking tool of the GOLD suite.43
For each compound, 25 independent docking runs were performed
and searching was conducted within a user-specified docking sphere
with the Genetic Algorithm protocol and the GoldScore scoring
function. The resulting docking complexes (ligand and side-chain
residues within 4.5 Å) were subjected to a MMFF94x energy
minimization until the rms conjugate gradient was <1 kcal mol −1 Å−1.
Prediction of antagonist−receptor complex stability (in terms of
corresponding pKi value) and the quantitative analysis for nonbonded
intermolecular interactions (H-bonds, transition metals, water bridges,
hydrophobic, electrostatic) were calculated and visualized using several
tools implemented in the MOE suite.41 Electrostatic and hydrophobic
contributions to the binding energy of individual amino acids have
been calculated with MOE.41 To estimate the electrostatic contribu-
tions, atomic charges for the ligands were calculated with MOPAC42
and the PM3/ESP methodology, whereas partial charges for the
protein amino acids were computed with the AMBER99 force field.
Molecular Dynamics. Each ligand−protein complex was embedded
in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid
bilayer (75 × 75 Å wide) and placed into the membrane according to
the suggested orientation reported in the “Orientations of Proteins in
Membranes (OPM)” database46 for the hA2A AR. The so-obtained
membrane−receptor complexes were solvated with TIP3P47 water using
the program Solvate 1.048 and neutralized by Na+/Cl− counterions to
a final concentration of 0.154 M. Membrane molecular dynamics
simulations were carried out on a GPU cluster with the ACEMD
program49 using the CHARMM27 force field50 and periodic boundaries
conditions. Initial parameters for the ligands were derived from the
CHARMM general force field for organic molecules51 by using the
paramchem service52 and were subsequently optimized at the MP2/
6-31G* level of theory53 (consistently with the CHARMM27 force field
parametrization) by using Gaussian 0954 and the implemented
parametrization tools in the VMD engine.55
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by a grant from the Italian Ministry
for University and Research (MIUR, FIRB RBNE03YA3L
project). The molecular modeling work coordinated by S.M.
has been carried out also with financial support from the
University of Padova, Italy. S.M. is also very grateful to
Chemical Computing Group and Acellera for the scientific and
technical partnership.
ABBREVIATIONS USED
■
AR, adenosine receptor; ATP, adenosine triphosphate; BM,
binding mode; CHO, Chinese hamster ovary; DMSO, dimethyl
sulfoxide; EDTA, ethylenediaminetetraacetic acid; GPCR, G
protein-coupled receptor; [3H]-CCPA, [3H]-2-chloro-6-cyclo-
pentyladenosine; IEFs, interaction energy fingerprints; MD,
molecular dynamics; NECA, 5′-N-ethylcarboxamidoadenosine;
POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
PTP, pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine; R-PIA, R-
N6-phenylisopropyladenosine; TLC, thin layer chromatogra-
phy; ZM241385, 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-
a][1,3,5]triazin-5-yl-amino]ethylphenol; SAR, structure−activ-
ity relationship; TM, transmembrane; EL2, second extracellular
loop
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