Novel diamino derivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selective adenosine A2a receptor antagonists

Article abstract of DOI:10.1021/jm0498396

Piperazine derivatives of 2-furanyl[1,2,4]triazolo[1,5-a][1,3,5]triazine have recently been demonstrated to be potent and selective adenosine A _2a receptor antagonists with oral activity in rodent models of Parkinson's disease. We have replaced

Full text of DOI:10.1021/jm0498396

J. Med. Chem. 2005, 48, 2009-2018
2009
Novel Diamino Derivatives of [1,2,4]Triazolo[1,5-a][1,3,5]triazine as Potent and
Selective Adenosine A2a Receptor Antagonists
Chi B. Vu,* Deborah Pan, Bo Peng, Gnanasambandam Kumaravel, Glenn Smits, Xiaowei Jin, Deepali Phadke,
Thomas Engber, Carol Huang, Jennifer Reilly, Stacy Tam, Donna Grant, Gregg Hetu, and Russell C. Petter
Department of Medicinal Chemistry, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142
Received February 24, 2004
Piperazine derivatives of 2-furanyl[1,2,4]triazolo[1,5-a][1,3,5]triazine have recently been
demonstrated to be potent and selective adenosine A2a receptor antagonists with oral activity
in rodent models of Parkinson’s disease. We have replaced the piperazinyl group with a variety
of linear, monocyclic, and bicyclic diamines. Of these diamines, (R)-2-(aminomethyl)pyrrolidine
is a particularly potent and selective replacement for the piperazinyl group. With this diamine
component, we have been able to prepare numerous analogues with low nanomolar affinity
toward the A2a receptor and good selectivity with respect to the A1 receptor (>200-fold in some
cases). Selected analogues from this series of [1,2,4]triazolo[1,5-a][1,3,5]triazine have now been
shown to be orally active in the mouse catalepsy model.
Introduction
receptor antagonists are also being extensively studied
by other investigators.12 Among the standard reference
compounds not derived from xanthine, compound 2
(SCH-58261) and compound 3 (ZM-241385) are two of
the most widely used (Figure 1).13,14 Originally disclosed
in the early 1990s, both of these compounds are fairly
selective toward A2a, although both suffered other
critical drawbacks such as a lack of oral bioavailability.15
Much work has taken place since then to improve the
pharmacological properties of these compounds, and it
appears that orally active compounds can now be
obtained using these core structures.16 Our laboratory
has studied extensively the [1,2,4]triazolo[1,5-a][1,3,5]-
triazine core structure of compound 3. We recently
demonstrated that piperazine derivatives of type 4 were
selective and potent adenosine A2a receptor antagonists
with oral efficacy in rodent models of Parkinson’s
disease.17 As an extension of this series, we began to
explore the possibility of replacing the piperazine with
the hope of retaining the A2a binding affinity and
selectivity over other adenosine receptors while improv-
ing the pharmacokinetic properties.
Our synthetic approach to this problem was inspired
by earlier work in the quinolone antibacterials. One of
the major advances in the quinolone research occurred
in the 1980s with the introduction of norfloxacin and
ciprofloxacin, both of which contained a critical pipera-
zine moiety.18 In later generations of quinolone anti-
biotics, the piperazine is replaced by a variety of
diamines. This type of structural modification allowed
the in vitro potency to be retained, and in some cases,
an improvement in pharmacokinetic properties was
observed. With our current lead series 4, we felt that a
systematic scanning of different diamines could be just
as fruitful. We selected compound 5 as a lead to improve
upon since its modest binding affinity toward A2a of 180
nM would allow us to definitively identify superior
replacements for the piperazine. To isolate the influence
of the diamines, we fixed the methyl isoxazole capping
group and the 2-furanyl[1,2,4]triazolo[1,5-a][1,3,5]triazine
moiety.
Adenosine has long been recognized as an important
mediator of numerous biological functions both in the
nervous system and in the peripheral tissues. Adenosine
can elicit very specific physiological responses when it
interacts with a particular adenosine receptor. The
adenosine receptors have been divided into four differ-
ent subtypes (A1, A2a, A2b, and A3),1 and selective
interaction with each of these receptors through the use
of agonists2 or antagonists3 has become an increasingly
attractive paradigm for therapeutic intervention. The
adenosine A2a receptors are widely expressed in the
basal ganglia.4 Modulation of these receptors, via the
action of selective A2a receptor agonists or antagonists,
will produce fairly significant changes in motor func-
tion.5 Human Parkinson’s disease is a motor disorder
that arises from the progressive loss of dopaminergic
nigrostriatal neurons of the basal ganglia.6 There are
currently a number of pharmacological models that can
recapitulate many of the symptoms displayed in Par-
kinsonian patients such as bradykinesia, tremor, and
rigidity, and A2a receptor antagonists appeared to have
a beneficial effect in many of these models.5,7-9 For
instance, in rats, catalepsy is a behavioral condition that
shares some similarity to human Parkinson’s disease.10
Fairly sustained catalepsy can be induced in rats by the
use of a selective A2a receptor agonist or a dopamine
antagonist such as haloperidol or reserpine.8 Adminis-
tration with selective A2a receptor antagonists has been
known to reverse this cataleptic condition in rats very
effectively.8 In marmosets, MPTP and a combination of
L-dopa and benserazide can be used to induce motor
disabilities that are comparable to patients with Par-
kinson’s disease.9 Oral administration with a selective
A2a receptor antagonist to these marmosets has recently
been shown to reverse motor disability in a dose-
dependent manner.9
Compound 1 (KW-6002) is a xanthine-based adeno-
sine A2a receptor antagonist.8,11 Nonxanthine-based A2a
* To whom correspondence should be addressed. Tel: 617-679-3861.
Fax: 617-679-2616. E-mail: chi.vu@biogenidec.com.
10.1021/jm0498396 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/12/2004

2010 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Vu et al.
with a 4-aminopiperidine group as in 17, a loss in A2a
binding affinity was observed. That loss in A2a activity
was even more pronounced when 3-aminopiperidine was
used to replace the piperazine (compound 18). A similar
loss in A2a activity was also observed when 4-(amino-
methyl)piperidine was used (compound 19). Interest-
ingly, when a 3-(aminomethyl)piperidine group was
used to replace the piperazine, as in 20, no significant
loss in A2a activity was observed. We then turned our
attention to the pyrrolidine ring. With 3-aminopyrroli-
dine, there was a fairly significant loss in A2a activity
(compound 21). However, when (R)-2-(aminomethyl)-
pyrrolidine was used, as in 22, a fairly significant
increase in A2a binding affinity was observed. Interest-
ingly, the (S)-isomer of 2-(aminomethy)pyrrolidine (com-
pound 23) was not as active as the (R)-isomer. Adding
an extra methyl group as in 24 also increased the
binding affinity toward A2a, and when the pyrrolidine
ring was changed to a piperidine ring as in 25, a
comparable A2a activity was observed.
After having identified (R)-2-(aminomethyl)pyrroli-
dine as a good replacement for the piperazine, we then
turned our attention to optimizing the capping group.
Table 2 lists analogues with substituted benzyl capping
groups, and Table 3 lists analogues with a variety of
heterocycles as capping groups. In general, analogues
prepared using the (R)-2-(aminomethy)pyrrolidine were
more active against A2a than those prepared using
piperazine as the diamine component.12 As shown in
Tables 2 and 3, most of the analogues that we prepared
had a Ki value of less than 50 nM against the adenosine
A2a receptor. The SAR, however, was slightly different
with these (R)-2-(aminomethyl)pyrrolidine derivatives.
As illustrated in compounds 26-28, meta substitution
appeared to be more favorable than either ortho or para
substitution and compound 27 had a Ki of 14 nM against
the A2a receptor. With the piperazine series, ortho
substitution was better than either meta or para
substitution.17 For comparison purposes, a small set of
piperazine derivatives of the general structure 4 is
shown in Table 4 (compounds 57-60). Even though the
meta substitution appeared to be favorable, having two
chloro substituents, as in 29, resulted in a decrease in
A2a binding affinity. Having two chloro substituents at
the ortho and para positions, as in 30, was, however,
comparable to one chloro substituent at the meta
position, and a Ki of 11 nM against A2a was obtained.
For compound 30, the Ki for the adenosine A1 receptor
was determined to be 1700 nM (>150-fold selectivity
between A2a and A1 receptors). As shown later on with
different analogues, many compounds within this series
of (R)-2-(aminomethyl)pyrrolidine displayed a very high
level of selectivity over the adenosine A1 receptor. The
methyl and fluoro substituents were comparable to the
chloro substituent in terms of A2a activity (compare
compounds 31 and 32 with compound 27). With com-
pound 33, when two fluorine atoms were installed at
the meta positions, a slight loss of A2a activity was
observed. This was similar to the pattern observed
above with the chloro derivative 27. Moving one of the
fluorine atoms from the meta position to the ortho
position, as in 34, did not result in any significant
changes in A2a activity. The ortho, para substitution
pattern shown in compound 35 was slightly more
Figure 1.
Results and Discussion
Table 1 lists the diamines used to replace the pipera-
zine group. Schemes 1 and 2 describe how these deriva-
tives were prepared. For symmetrical amines, such as
the ones used to prepare compounds 9-11 and 13-16,
an excess of the unprotected diamine could be condensed
directly onto sulfone 6 (Scheme 1).14 Purification by
column chromatography removes the unreacted di-
amine, and then, alkylation with the mesylate deriva-
tive of (5-methyl-isoxazol-3-yl)methanol afforded the
desired derivatives. For unsymmetrical amines such as
the ones used to prepare compounds 12 and 17-25, the
BOC-protected diamines were used. The BOC-protected
diamines were commercially available with the excep-
tion of the two used to prepare compounds 24 and 25.
These two diamines were prepared by taking the
appropriate BOC-protected amino acids and converting
them to the corresponding amides through the reaction
with methylamine in the presence of EDC/HOBT and
N-methylmorpholine. These amides were then reduced
to the desired N-methyl amines using BH3‚THF.
As shown in Table 1, homopiperazine (compound 9)
was fairly similar to piperazine and a slight gain of A2a
binding affinity was observed. The ethylenediamine
linker represented another obvious starting point, and
only a modest loss in A2a binding affinity was observed
(compound 10). Methylating the amino group as in 11
and 12 did improve the A2a binding. Replacing the
ethylenediamine linker with a longer chain as in 13 and
14 resulted in a substantial loss of A2a binding affinity.
A substantial loss of A2a binding affinity was also
observed when the piperazino group was replaced with
the more constrained amines such as those shown in
15 and 16. When the piperazino group was replaced

Novel Diamino Derivatives of Triazolotriazine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2011
Table 1. Variation of the Diamino Portion of Substituted [1,2,4]Triazolo[1,5-a][1,3,5]triazinesa
a For the A2a receptor, the membranes were prepared from rat brain tissues and the radioligand binding assay was performed using
the radioligand [3H]ZM-241385. All Ki values were calculated from binding curves generated from the mean of three determinations per
concentration, with the variation in individual values of <15%.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) Symmetrical diamine, CH3CN,
reflux (for example, piperazine and homopiperazine were used,
respectively, in the preparation of 5 and 9). (b) Mesylate of (5-
methyl-isoxazol-3-yl)methanol, Et3N, CH2Cl2, room temperature.
a Reagents and conditions: (a) BOC-protected diamine, CH3CN,
Et3N, reflux (for example, in the preparation of 17, 4-amino-1-
BOC-piperidine was used). (b) 25% TFA, 75% CH2Cl2, room
temperature. (c) Mesylate of (5-methyl-isoxazol-3-yl)methanol,
Et3N, CH2Cl2, room temperature.
effective, and a Ki of 14 nM against A2a was obtained.
The more effective difluoro substitution patterns were
either ortho, meta (compound 36) or ortho, ortho (com-
pound 38) where low nanomolar binding affinity toward
A2a was observed. Because the chloro substituent was
comparable to the fluoro substituent, compound 37 was
essentially equivalent to 36. Adding one more fluoro
substituent as in 39-41 was still acceptable, and fairly
potent compounds could be obtained. Compound 41, in
particular, was not only very potent against A2a (Ki ) 2
nM)but also very selective over the A1 receptor (400-
fold selective). When one of the fluoro groups was

2012 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Table 2. Benzyl Capping Groupsa
Vu et al.
a For the A2a receptor, the membranes were prepared from rat brain tissues and the radioligand binding assay was performed using
the radioligand [3H]ZM-241385. For the A1 receptor, the membranes were prepared from rat cerebral cortex and the radioligand binding
assay was performed using the radioligand [3H]DPCPX. All Ki values were calculated from binding curves generated from the mean of
three determinations per concentration, with the variation in individual values of <15%.
replaced with a trifluoromethyl group, as in compounds
42, 43, and 45, no significant loss of A2a binding affinity
was observed and fairly potent compounds could still
be obtained. For comparison, we have also determined
the A2a and A1 binding affinities for a number of
reference compounds. In our radioligand binding assay,
we used compound 2 as the control compound. The A2a
Ki for compound 2 was 37 nM, and the A1 Ki was 390
nM. As additional points of reference, the binding
affinities for compounds 1 and 3 were determined. For
compound 1, the A2a Ki was 42 nM and the A1 Ki was
930 nM. For compound 3, the A2a Ki was 0.9 nM and
the A1 Ki was 680 nM. This clearly indicated that in
our preliminary round of optimization, we had identified
compounds with better A2a binding affinity than those
displayed with compounds 1 and 3. The potency and
selectivity level of compound 41 were comparable to
those of compound 3, one of the most potent and
selective adenosine A2a antagonists disclosed to date.
With heterocyclic capping groups, 3-substitution,
again, appears to be slightly more favorable than
2-substitution. This was first illustrated with the three
pyridinyl analogues (compounds 46-48). Having two
methyl groups as in 49 certainly was not as favorable
as having one methyl group at the 3-position, as in
compound 22. When a halogen was substituted onto a
heterocyclic ring as in 50, an increase in A2a activity
was observed. A similar increase in A2a activity was also
observed with the two furans 51 and 52 (again, note
the increase in A2a activity when the chlorine atom was
installed at the appropriate position on the capping
furan ring). Perhaps a more dramatic illustration of the
use of the 3-substitution pattern is illustrated with
compounds 53 and 54. Clearly, having two chlorine

Novel Diamino Derivatives of Triazolotriazine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2013
Table 3. Heterocyclic Capping Groupsa
Table 4. Piperazine Derivatives of
[1,2,4]Triazolo[1,5-a][1,3,5]triazinea
a Taken in part from ref 17. Refer to Table 2 for membrane
preparation and details regarding the radioligand binding assay.
previously (additional details could also be found in the
Experimental Section below). The ease of observation
and the speed of the response make this assay a
convenient and relatively high-throughput screen of in
vivo activity.
All of the compounds listed in Tables 2 and 3 were
tested in this mouse catalepsy model. The xanthine
derivative 1 was employed as the positive control. A full
dose-response curve for compound 1 was determined,
and its ED50 was 1 mg/kg p.o. All 34 new analogues
shown in Tables 2 and 3 were tested in the mouse
catalepsy model, and eight of these showed good oral
activity at 10 mg/kg: 30, 33, 35, 38, 41, 43, 52, and 54.
Oral activity at 3 mg/kg was not observed with any of
these (R)-2-(aminomethyl)pyrrolidine analogues. This is
in sharp contrast to the piperazine series 4 where oral
activity at 3 mg/kg was observed in the mouse catalepsy
model for a number of analogues. Analogues derived
from (R)-2-(aminomethyl)pyrrolidine in general ap-
peared to be highly potent and selective in vitro but
were not as potent in vivo as those derived from
piperazine. Figures 2 and 3 clearly show that the
piperazine analogue 60 and compound 1 were superior
to analogues derived from (R)-2-(aminomethyl)pyrroli-
dine. In these studies, both 60 and 1 were dosed orally
at a lower dose of 3 mg/kg.
a Refer to Table 2 for membrane preparation and details
regarding the radioligand binding assay. All Ki values were
calculated from binding curves generated from the mean of three
determinations per concentration, with the variation in individual
values of <15%.
On the basis of the sustained oral activity at the 120
min time point, compound 38 was selected for additional
profiling in the rat. In the rat catalepsy model, the full
dose-response for 38 was determined and its ED50 was
10 mg/kg when administered orally. The pharmacoki-
netic properties of 38 were also determined using
standard protocols. Here, rats were dosed both IV and
orally at 1 mg/kg and plasma samples were taken at
nine time points over 24 h. Compound 38 did not appear
to be orally bioavailable since no parent compound was
detected in the plasma (%F ) 0%). Compound 38,
atoms at the 2- and 6-positions was not as potent as
having just one chlorine atom at the 3-position. Finally,
slightly larger capping groups, such as benzofuran and
quinoline, were still acceptable and fairly potent ana-
logues could still be obtained (compounds 55 and 56).
For an initial evaluation of both oral bioavailability
and CNS activity, we used a mouse catalepsy model.17
This is a well-established efficacy model for Parkinson’s
disease in which catalepsy is induced by haloperidol. A
detailed description of this model has been disclosed

2014 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Vu et al.
selective adenosine A2a receptor antagonists disclosed
to date. Even though some of these new [1,2,4]triazolo-
[1,5-a]triazines have now been shown to be orally active
in rodent models for Parkinson’s disease, further opti-
mization is needed to improve their oral bioavailability.
These results will be disclosed in due course.
Experimental Section
General Information. All proton nuclear magnetic reso-
nance spectra were determined in the indicated solvent using
a 400 MHz Bruker with the appropriate internal standard.
Low-resolution MS were performed on a Micromass/single
quadrupole/electrospray platform. High-resolution MS were
performed on a MALDI-TOF MS (Voyager-DE STR, Perseptive
Biosystems) in the reflector mode with delayed extraction and
an accelerating voltage of 20 kV. Each spectrum was an
average of 100 laser shots, and the experimental monoisotopic
M+ + H value was calculated by averaging five spectra.
Elemental analyses were carried out at Quantitative Tech-
nologies Inc. (QTI, Whitehouse, NJ). Unless indicated other-
wise, reagent grade chemicals and solvents were purchased
from Aldrich, Lancaster, Fisher, or Maybridge. Analytical
HPLC analysis was carried out using a HP 1100 series, with
a 100 mm × 4.6 mm ID YMC column with S-5 µm packing
(catalog no. AM-301). Preparative HPLC was carried out using
a Gilson Platform equipped with UV/visible detector and an
automatic fraction collector. Preparative HPLC columns were
50 mm × 20 mm IC YMC column with S-5 µm packing. HPLC
solvents (H2O and CH3CN) were buffered with 0.1% TFA.
2-Furan-2-yl-5-[4-(5-methyl-isoxazol-3-ylmethyl)[1,4]-
diazepan-1-yl][1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
ylamine (9). In a typical procedure, 0.7 mmol of 2-furan-2-
yl-5-methanesulfonyl[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-
ylamine (6)14 was suspended in 10 mL of CH3CN along with 5
equiv of homopiperazine. The reaction mixture was stirred
under reflux for 2 h. It was then cooled to room temperature
and concentrated under reduced pressure. The residue was
taken up in CH2Cl2 and washed with H2O and brine, dried
(Na2SO4), and concentrated under reduced pressure. The
resulting crude product was purified by column chromatog-
raphy (SiO2, 95% CH2Cl2, 4% MeOH, 1% Et3N) to afford the
intermediate homopiperazine.
Figure 2. Mouse catalepsy data. For the mouse catalepsy
study, CD-1 mice (25-30 g) were injected subcutaneously with
3 mg/kg of haloperidol in order to induce catalepsy. Compounds
30, 33, 35, and 38 were dosed orally at 10 mg/kg. Compound
60 was dosed orally at 3 mg/kg. All test compounds were
formulated as the hydrochloride salt and dissolved in saline
before administration to the animals. Catalepsy was measured
at four time points over a period of 2 h. The data represent
the mean of six animals per group. Catalepsy was measured
as the time in seconds until the animal removed at least one
forepaw from the bar, with a maximum value of 120 s per test.
Values for each animal in the drug-treated groups were
expressed as the percentage of the mean value for the vehicle-
treated control group at that time point. These values are
shown on the y-axis.
In a separate flask, (5-methyl-isoxazol-3-yl)methanol (32 mg,
0.28 mmol) was dissolved in 4 mL of CH2Cl2 along with 1.3
equiv of Et3N. The solution was cooled in an ice bath, and
methanesulfonyl chloride (1.2 equiv) was added. The reaction
mixture was warmed to room temperature and stirred for 45
min. It was then quenched with brine, and the two layers were
separated. The organic layer was dried (Na2SO4) and concen-
trated under reduced pressure to afford the mesylate deriva-
tive. This mesylate was then added to a solution containing
0.14 mmol of the homopiperazine derivative prepared above
and Et3N (0.3 mmol) in 3 mL of CH3CN. The resulting reaction
mixture was stirred at room temperature for 18 h. It was then
concentrated and purified by preparative HPLC using a
mixture of aqueous CH3CN that has been buffered with 0.1%
TFA. Spectroscopic data for 9: 1H NMR (DMSO-d6): δ 8.20
(br s, 2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 7.00 (d, J ) 3.6 Hz, 1 H),
6.68 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H), 4.20 (br s, 2
H), 3.60-2.70 (m, 10 H), 2.35 (s, 3H) ppm. MS m/z ) 396 amu
(M+ + H). Anal. (C18H21N9O2) C, H, N.
Figure 3. Mouse catalepsy data. Refer to Figure 2 for
additional details regarding the mouse catalepsy study. Com-
pounds 41, 43, 52, and 54 were dosed orally at 10 mg/kg.
Compound 1 was dosed orally at 3 mg/kg.
however, still had a reasonably long IV half-life of 1.7
h and a fairly large volume of distribution of 7 L/kg.
Future work in this series will focus on the determina-
tion of active metabolites that may be responsible for
the observed efficacy in the catalepsy models. In addi-
tion, modifications to the basic [1,2,4]triazolo[1,5-a]-
triazine core structure will be carried out in order to
improve the overall pharmacological properties of these
(R)-2-(aminomethyl)pyrrolidines.
In summary, we have shown how the piperazine of 4
could be replaced effectively with a number of different
diamines without experiencing any loss in in vitro
potency. Among the diamines that were examined, (R)-
2-(aminomethyl)pyrrolidine emerged as a particularly
effective replacement. The basic [1,2,4]triazolo[1,5-a]-
triazine core structure could now be modified effectively
by using the appropriately substituted (R)-2-(amino-
methyl)pyrrolidines. An initial round of optimization
has resulted in the discovery of a number of potent and
selective adenosine A2a receptor antagonists. In in vitro
binding assays, compound 41, for example, was about
as active as compound 3, one of the most potent and
General Procedure for the Preparation of Compounds
10, 11, and 13-16. The same procedure outlined in the
preparation of compound 9 was used to prepare compounds
10, 11, and 13-16. The appropriate symmetrical diamine was
used in each case (see examples below).
N5-(2-(N-Methyl-N-((5-methylisoxazol-3-yl)methyl)ami-
no)ethyl)-2-(furan-2-yl)-N5-methyl[1,2,4]triazolo[1,5-a]-
[1,3,5]triazine-5,7-diamine (11). The same procedure out-
lined in the preparation of compound 9 was used to prepare
compound 11. N,N′-Dimethylethylenediamine was used in-
stead of homopiperazine. Spectroscopic data for 11: 1H NMR
(DMSO-d6): δ 8.30 (br s, 2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 6.90

Novel Diamino Derivatives of Triazolotriazine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2015
(d, J ) 3.6 Hz, 1 H), 6.50 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s,
1 H), 4.60 (br s, 2 H), 3.60-3.10 (m, 4 H), 2.75 (s, 3 H), 2.35 (s,
3H), 2.25 (s, 3H) ppm. MS m/z ) 384 amu (M+ + H). High-
resolution MS (M+ + H) calcd for C17H22N9O2, 384.1896; found,
384.1885. HPLC retention time ) 3.58 min (HP1100 analytical
HPLC, gradient elution at a rate of 2 mL/min, 5% aqueous
CH3CN to 95% aqueous CH3CN in 10 min).
2-(Furan-2-yl)-5-(5-((5-methylisoxazol-3-yl)methyl)-2,5-
diaza-bicyclo[2.2.1]heptan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]-
triazin-7-amine (16). The same procedure outlined in the
preparation of compound 9 was used to prepare compound 16.
Commercially available (1S,4S)-(+)-2,5-diazabicyclo[2.2.1]-
heptane was used instead of homopiperazine. Spectroscopic
data for 16: 1H NMR (DMSO-d6): δ 8.20 (br s, 2 H), 7.80 (d,
J ) 1.0 Hz, 1 H), 7.00 (d, J ) 3.6 Hz, 1 H), 6.60 (dd, J ) 3.6
Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H), 4.80 (br s, 2 H), 4.20-3.20 (m,
8 H), 2.35 (s, 3H) ppm. MS m/z ) 394 amu (M+ + H). High-
resolution MS (M+ + H) calcd for C18H20N9O2, 394.1740; found,
394.1720. HPLC retention time ) 3.23 min (HP1100 analytical
HPLC, gradient elution at a rate of 2 mL/min, 5% aqueous
CH3CN to 95% aqueous CH3CN in 10 min).
3.6 Hz, 1 H), 6.60 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H),
4.50 (br s, 2 H), 3.60-1.80 (m, 9 H), 2.35 (s, 3H) ppm. MS m/z
) 396 amu (M+ + H). High-resolution MS (M+ + H) calcd
for C18H20N9O2, 396.1896; found, 396.1900. HPLC retention
time ) 3.37 min (HP1100 analytical HPLC, gradient elution
at a rate of 2 mL/min, 5% aqueous CH3CN to 95% aqueous
CH3CN in 10 min).
2-(Furan-2-yl)-N5-(1-((5-methylisoxazol-3-yl)methyl)pi-
peridin-3-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (18). The same procedure outlined above in the
preparation of compound 22 was used. However, racemic
3-amino-1-BOC-piperidine was used instead of (R)-2-amino-
methyl-1-BOC-pyrrolidine. Spectroscopic data for 18: 1H NMR
(DMSO-d6): δ 8.30 (br s, 2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 6.90
(d, J ) 3.6 Hz, 1 H), 6.60 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s,
1 H), 4.50 (br s, 2 H), 3.60-1.80 (m, 9 H), 2.35 (s, 3H) ppm.
MS m/z ) 396 amu (M+ + H). High-resolution MS (M+ + H)
calcd for C18H20N9O2, 396.1896; found, 396.1885. HPLC reten-
tion time ) 3.46 min (HP1100 analytical HPLC, gradient
elution at a rate of 2 mL/min, 5% aqueous CH3CN to 95%
aqueous CH3CN in 10 min).
2-(Furan-2-yl)-N5-(((R)-1-((5-methylisoxazol-3-yl)meth-
yl)pyrrolidin-2-yl)methyl)[1,2,4]triazolo[1,5-a][1,3,5]-
triazine-5,7-diamine (22). In a typical procedure, 2.5 mmol
of 2-furan-2-yl-5-methanesulfonyl[1,2,4]triazolo[1,5-a]triazin-
7-ylamine (6)14 was suspended in 20 mL of CH3CN along with
5 mmol of (R)-2-aminomethyl-1-BOC-pyrrolidine (commercially
available from Astatech). The reaction mixture was stirred
under reflux for 2 h. It was then cooled to room temperature
and concentrated under reduced pressure. The resulting
residue was diluted with CH2Cl2 and washed with dilute 1%
aqueous citric acid and brine, dried (Na2SO4), and concentrated
under reduced pressure. Purification by chromatography (98%
CH2Cl2, 2% MeOH) afforded 880 mg of the BOC-protected
material. This material was dissolved in 6 mL of 25% TFA in
CH2Cl2 and allowed to stand at room temperature for 2 h. The
reaction mixture was concentrated under reduced pressure to
afford the TFA salt of 2-furan-2-yl-N5-pyrrolidin-2-ylmethyl-
[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine.
In a separate flask, (5-methyl-isoxazol-3-yl)methanol (32 mg,
0.28 mmol) was dissolved in 4 mL of CH2Cl2 along with 1.3
equiv of Et3N. The solution was cooled in an ice bath, and
methanesulfonyl chloride (1.2 equiv) was added. The reaction
mixture was warmed to room temperature and stirred for 45
min. It was then quenched with brine, and the two layers were
separated. The organic layer was dried (Na2SO4) and concen-
trated under reduced pressure to afford the mesylate deriva-
tive. This mesylate was then added to a solution containing
0.15 mmol of the pyrrolidine salt prepared above and Et3N
(0.5 mmol) in 3 mL of CH3CN. The resulting reaction mixture
was stirred at room temperature for 18 h. It was then concen-
trated and purified by preparative HPLC using a mixture of
aqueous CH3CN that had been buffered with 0.1% TFA.
Spectroscopic data for 22: 1H NMR (DMSO-d6): δ 8.20 (br s,
2 H), 7.85 (d, J ) 1.0 Hz, 1 H), 7.00 (d, J ) 3.6 Hz, 1 H), 6.60
(dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H), 4.20 (br s, 2 H),
3.60-3.10 (m, 9 H), 2.35 (s, 3H) ppm. MS m/z ) 396 amu (M+
+ H). High-resolution MS (M+ + H) calcd for C18H22N9O2,
396.1896; found, 396.1900. HPLC retention time ) 3.63 min
(HP1100 analytical HPLC, gradient elution at a rate of 2
mL/min, 5% aqueous CH3CN to 95% aqueous CH3CN in 10
min).
2-(Furan-2-yl)-N5-(1-((5-methylisoxazol-3-yl)methyl)-
pyrrolidin-3-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (21). The same procedure outlined above in the
preparation of compound 22 was used. However, (R)-3-amino-
1-BOC-pyrrolidine was used instead of (R)-2-aminomethyl-1-
BOC-pyrrolidine. Spectroscopic data for 21: 1H NMR (DMSO-
d6): δ 8.20 (br s, 2 H), 7.85 (d, J ) 1.0 Hz, 1 H), 7.00 (d, J )
3.6 Hz, 1 H), 6.60 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H),
4.40 (br s, 2 H), 3.60-3.10 (m, 7 H), 2.35 (s, 3H) ppm. MS m/z
) 382 amu (M+ + H). High-resolution MS (M+ + H) calcd
for C17H20N9O2, 382.1740; found, 382.1726. HPLC retention
time ) 3.49 min (HP1100 analytical HPLC, gradient elution
at a rate of 2 mL/min, 5% aqueous CH3CN to 95% aqueous
CH3CN in 10 min).
2-(Furan-2-yl)-N5-methyl-N5-(((R)-1-((5-methylisoxazol-
3-yl)methyl)pyrrolidin-2-yl)methyl)[1,2,4]triazolo[1,5-a]-
[1,3,5]triazine-5,7-diamine (24). The same procedure out-
lined above in the preparation of compound 22 was used.
However, (R)-2-methylaminomethyl-1-BOC-pyrrolidine was
used instead of (R)-2-aminomethyl-1-BOC-pyrrolidine. (R)-2-
Methylaminomethyl-1-BOC-pyrrolidine was prepared as fol-
lows: (R)-BOC-proline (4.8 g, 22.3 mmol) was suspended in
100 mL of THF. EDC (5.13 g, 1.2 equiv) was added, followed
by HOBT (3.62 g, 1.2 equiv) and N-methylmorpholine (3.7 mL,
1.5 equiv). The reaction mixture was stirred at room temper-
ature for 30 min, and 35 mL of methylamine in THF (2.0 M
solution in THF, 3 equiv) was added. The resulting reaction
mixture was stirred at room temperature for 18 h. It was then
concentrated under reduced pressure, and the residue was
taken up in CH2Cl2. This solution was washed with diluted
NaHCO3, water, dilute 5% aqueous citric acid, and brine, dried
(Na2SO4), and concentrated to afford 4.8 g of the crude
carboxamide. This material was dissolved in 100 mL of
anhydrous THF and cooled to 0 °C. A solution of borane-THF
(53 mL of the 1.0 M solution in THF, 2.5 equiv) was added,
and the reaction mixture was allowed to warm to room
temperature on its own and stirred at room temperature for
18 h. It was then cooled to 0 °C and carefully quenched with
50 mL of methanol. The reaction mixture was concentrated
under reduced pressure. The resulting residue was redissolved
in 50 mL of methanol and 100 mL of EtOAc and concentrated
under reduced pressure. This trituration with MeOH/EtOAc
and concentration were repeated three more times to afford
essentially a quantitative yield of (R)-2-methylaminomethyl-
1-BOC-pyrrolidine. Spectroscopic data for 24: 1H NMR (DMSO-
d6): δ 8.20 (br s, 2 H), 7.85 (d, J ) 1.0 Hz, 1 H), 7.00 (d, J )
3.6 Hz, 1 H), 6.60 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 6.30 (s, 1 H),
4.40 (br s, 2 H), 3.60-3.10 (m, 9 H), 2.90 (s, 3 H), 2.35 (s, 3H)
ppm. MS m/z ) 410 amu (M+ + H). High-resolution MS (M+
+ H) calcd for C19H24N9O2, 410.2053; found, 410.2022. HPLC
retention time ) 3.86 min (HP1100 analytical HPLC, gradient
elution at a rate of 2 mL/min, 5% aqueous CH3CN to 95%
aqueous CH3CN in 10 min).
General Procedure for the Preparation of Compounds
12, 17-21, and 23. The same procedure outlined above in the
preparation of compound 22 was used to prepare compounds
12, 17-21, and 23. The appropriate BOC-protected diamine
was used in each case (see examples below).
2-(Furan-2-yl)-N5-(1-((5-methylisoxazol-3-yl)methyl)pi-
peridin-4-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (17). The same procedure outlined above in the
preparation of compound 22 was used. However, 4-amino-1-
BOC-piperidine was used instead of (R)-2-aminomethyl-1-
BOC-pyrrolidine. Spectroscopic data for 17: 1H NMR (DMSO-
d6): δ 8.30 (br s, 2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 6.90 (d, J )

2016 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Vu et al.
N5-(((R)-1-(2-chlorobenzyl)pyrrolidin-2-yl)methyl)-2-
(furan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (26). The TFA salt of 2-furan-2-yl-N5-pyrrolidin-2-
ylmethyl[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine, pre-
pared as outlined above in the preparation of compound
22, was used as the starting material. This TFA salt (2 mmol)
was converted to the corresponding free amine first by dis-
solving in 5 mL of water and neutralized with 1 molar
equivalent of aqueous NaOH. The resulting solution was
concentrated to dryness to afford the desired pyrrolidine as
a mixture of the free amine and sodium salt. This pyrroli-
dine mixture (0.15 mmol) was suspended in 4 mL of CH2Cl2
along with 2 equiv of 2-chlorobenzaldehyde and 25 µL of
glacial acetic acid. The resulting reaction mixture was stirred
at room temperature for 30 min, and sodium triacetoxyboro-
hydride (4 equiv) was added in a single portion. The resulting
reaction mixture was stirred at room temperature for 18 h. It
was then concentrated under a stream of N2 and purified by
preparative HPLC using a mixture of aqueous CH3CN that
had been buffered with 0.1% TFA. Analytically pure sam-
ples could also be obtained by column chromatography (SiO2)
using a mixture of 95% CH2Cl2 and 5% MeOH. Spectroscopic
data for 26: 1H NMR (DMSO-d6): δ 8.20 (br s, 2 H), 7.80 (d,
J ) 1.0 Hz, 1 H), 7.30-7.10 (m, 4 H), 7.00 (d, J ) 3.6 Hz, 1
H), 6.68 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 4.20 (br s, 2 H),
3.60-2.50 (m, 9 H) ppm. MS m/z ) 426 amu (M+ + H). Anal.
(C20H21ClN8O) C, H, N.
General Procedure for the Preparation of Compounds
27-45. The rest of the compounds shown in Table 2 (com-
pounds 27-45) were prepared using the same procedure
outlined above in the preparation of compound 26 using the
appropriate aldehyde (see examples below).
N5-(((R)-1-(3-Fluorobenzyl)pyrrolidin-2-yl)methyl)-2-
(furan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (32). The same procedure outlined above in the
preparation of compound 26 was used. However, 3-fluoroben-
zaldehyde was used instead of 2-chlorobenzaldehyde. Spec-
troscopic data for 32: 1H NMR (DMSO-d6): δ 8.20 (br s, 2 H),
7.80 (d, J ) 1.0 Hz, 1 H), 7.30-7.10 (m, 4 H), 7.00 (d, J ) 3.6
Hz, 1 H), 6.68 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 4.80 (br s, 2 H),
3.60-2.50 (m, 9 H) ppm. MS m/z ) 409 amu (M+ + H). Anal.
(C20H21FN8O) C, H, N.
N5-(((R)-1-(2,6-Difluorobenzyl)pyrrolidin-2-yl)methyl)-
2-(furan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (38). The same procedure outlined above in the
preparation of compound 26 was used. However, 2,6-difluo-
robenzaldehyde was used instead of 2-chlorobenzaldehyde.
Spectroscopic data for 38: 1H NMR (DMSO-d6): δ 8.20 (br s,
2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 7.60 (br s, 1 H), 7.28-7.22 (m,
1 H), 6.91-6.86 (m, 2 H), 7.00 (d, J ) 3.6 Hz, 1 H), 6.68 (dd,
J ) 3.6 Hz, 1.0 Hz, 1 H), 5.20 (br s, 2 H), 3.60-2.50 (m, 9 H)
ppm. MS m/z ) 427 amu (M+ + H). Anal. (C20H20F2N8O) C,
H, N
N5-(((R)-1-(2,4,6-Trifluorobenzyl)pyrrolidin-2-yl)methyl)-
2-(furan-2-yl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-di-
amine (41). The same procedure outlined above in the
preparation of compound 26 was used. However, 2,4,6-trifluo-
robenzaldehyde was used instead of 2-chlorobenzaldehyde.
Spectroscopic data for 41: 1H NMR (DMSO-d6): δ 8.20 (br s,
2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 6.91-6.86 (m, 2 H), 7.00 (d, J
) 3.6 Hz, 1 H), 6.68 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 5.20 (br s,
2 H), 3.60-2.50 (m, 9 H) ppm. MS m/z ) 427 amu (M+ + H).
Anal. (C20H19F3N8O‚1H2O) C, H, N.
Spectroscopic data for compound 46: 1H NMR (DMSO-d6): δ
8.5 (br s, 1 H), 8.20 (br s, 2 H), 7.80 (d, J ) 1.0 Hz, 1 H), 7.60
(br s, 1 H), 7.50-7.22 (m, 3 H), 7.00 (d, J ) 3.6 Hz, 1 H), 6.68
(dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 4.20 (br s, 2 H), 3.60-2.50 (m,
9 H) ppm. MS m/z ) 392 amu (M+ + H). Anal. (C19H21N9O) C,
H, N.
General Procedure for the Preparation of Compounds
49, 53, and 54. An alkylation procedure was used to prepare
these compounds. The preparation of 49 is shown below.
Compounds 53 and 54 were prepared using the same general
procedure outlined in the preparation of 49 using the ap-
propriate halide.
2-(Furan-2-yl)-N5-(((R)-1-((3,5-dimethylisoxazol-4-yl)-
methyl)pyrrolidin-2-yl)methyl)[1,2,4]triazolo[1,5-a][1,3,5]-
triazine-5,7-diamine (49). The TFA salt of 2-furan-2-yl-N5-
pyrrolidin-2-ylmethyl[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-
diamine, prepared as outlined above in the preparation of
compound 19, was used as the starting material. This TFA
salt (2 mmol) was converted to the corresponding free amine
first by dissolving in 5 mL of water and neutralized with 1
molar equivalent of aqueous NaOH. The resulting solution was
concentrated to dryness to afford the desired pyrrolidine as a
mixture of the free amine and NaCF3CO2H. This pyrrolidine
mixture (0.10 mmol) was suspended in 2 mL of CH3CN along
with 2 equiv of 4-chloromethyl-3,5-dimethyl-isoxazole and 3
equiv of Et3N. The resulting reaction mixture was stirred at
room temperature for 18 h and then concentrated under a
stream of N2. The resulting crude product was purified by
preparative HPLC. Spectroscopic data for 49: 1H NMR
(DMSO-d6): δ 8.20 (br s, 2 H), 7.85 (d, J ) 1.0 Hz, 1 H), 7.00
(d, J ) 3.6 Hz, 1 H), 6.60 (dd, J ) 3.6 Hz, 1.0 Hz, 1 H), 4.20
(br s, 2 H), 3.60-3.10 (m, 9 H), 2.35 (s, 3H), 2.10 (s, 3H) ppm
MS m/z ) 410 amu (M+ + H). HPLC retention time ) 3.10
min (HP1100 analytical HPLC, gradient elution at a rate of 2
mL/min, 5% aqueous CH3CN to 95% aqueous CH3CN in 10
min).
Formation of the Hydrochloride Salt for 30, 33, 35, 38,
41, 43, 52, 54, and 60. For each of these compounds, the
corresponding hydrochloride salt was prepared as follows.
Each compound (0.2 mmol) was dissolved in 10 mL of 1:1
CH3CN/H2O. Aqueous HCl (0.2 mL of a 1 N solution) was then
added, and the resulting solution was lyophilized to afford the
desired hydrochloride salt.
Biological Assays. 1. A2a Receptor. The membranes were
prepared from rat brain tissues purchased from Pel-Freez. The
tissues were homogenized in buffer A (10 mM EDTA, 10 mM
Na-HEPES, pH 7.4) supplemented with protease inhibitors
(10 µg/mL benzamidine, 100 µM PMSF, and 2 µg/mL each of
aprotinin, pepstatin, and leupeptin) and centrifuged at 20000g
for 20 min. The pellets were resuspended and washed twice
with buffer HE (10 mM Na-HEPES, 1 mM EDTA, pH 7.4,
plus protease inhibitors). The final pellets were resuspended
in buffer HE, supplemented with 10% (w/v) sucrose and
protease inhibitors, and frozen in aliquots at -80 °C. Protein
concentrations were measured using the BCA protein assay
kit (Pierce).
2. Rat A1 Receptor. The membranes were prepared from
rat cerebral cortex isolated from freshly euthanized rats. The
tissues were homogenized in buffer A (10 mM EDTA, 10 mM
Na-HEPES, pH 7.4) supplemented with protease inhibitors
(10 µg/mL benzamidine, 100 µM PMSF, and 2 µg/mL each of
aprotinin, pepstatin, and leupeptin) and centrifuged at 20000g
for 20 min. The pellets were resuspended and washed twice
with buffer HE (10 mM Na-HEPES, 1 mM EDTA, pH 7.4,
plus protease inhibitors). The final pellets were resuspended
in buffer HE, supplemented with 10% (w/v) sucrose and
protease inhibitors, and frozen in aliquots at -80 °C. The
protein concentrations were measured using the BCA protein
assay kit (Pierce).
General Procedure for the Preparation of Compounds
46-48, 50-52, and 55-56. These compounds were prepared
using the same reductive amination conditions that were
outlined above in the preparation of compound 26. The
appropriate heterocyclic aldehyde was used (see example
below).
2-(Furan-2-yl)-N5-(((R)-1-((pyridin-2-yl)methyl)pyrro-
lidin-2-yl)methyl)[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-
diamine (46). The same procedure outlined above in the
preparation of compound 26 was used. However, 2-pyridin-
ecarboxaldehyde was used instead of 2-chlorobenzaldehyde.
3. Radioligand Binding Assays. Membranes (40-70 µg
of membrane protein), radioligands, and varying concentra-
tions of competing ligands were incubated in triplicate in
0.1 mL of buffer HE plus 2 units/mL adenosine deaminase for
2.5 h at 21 °C. Radioligand [3H]DPCPX was used for competi-

Novel Diamino Derivatives of Triazolotriazine
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2017
tion binding assays on A1 receptors, and [3H]ZM241385 was
used for A2a adenosine receptors. Nonspecific binding was
measured in the presence of 10 µM NECA for A1 receptors or
10 µM XAC for A2A receptors. The binding assays were
terminated by filtration over Whatman GF/C glass fiber fil-
ters using a BRANDEL cell harvester. The filters were rinsed
three times with 3-4 mL of ice-cold 10 mM Tris-HCl, pH 7.4,
and 5 mM MgCl2 at 4 °C and were counted in a Wallac
â-counter.
4. Analysis of Binding Data. For Ki determinations,
competition binding data were fit to a single-site binding model
and plotted using Prizm GraphPad. The Cheng-Prusoff equa-
tion KI ) IC50/(1 + [I]/KD) was used to calculate KI values from
IC50 values, where KI is the affinity constant for the competing
ligand, [I] is the concentration of the free radioligand, and KD
is the dissociation constant for the radioligand.
5. Catalepsy Experiments. Haloperidol-induced catalepsy
was used to mimic the effects of Parkinson’s disease in
rats and mice. The animals were injected with haloperidol,
which causes immobility. A test compound was then admin-
istered orally, and the compound’s ability to reverse these
Parkinson’s-like symptoms was analyzed. All experiments
using animals were conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals and
approved by the Institutional Animal Care and Use Commit-
tee. The IACUC protocol number for all of the catalepsy studies
is 105-00.
6. Rats. Male Sprague-Dawley rats (225-275 g) were
injected with haloperidol (1 mg/kg s.c.). Every 30 min for the
next 3 h, catalepsy was measured using the bar test.21 In this
test, the rats’ forelimbs were placed on an aluminum bar (1
cm in diameter) suspended horizontally 10 cm above the
surface of the bench. The elapsed time until the rat places one
forepaw back on the bench is measured, with a maximum
time of 120 s allowed. Once rats show a stable baseline
cataleptic response (about 3 h after haloperidol injection), the
test compound or vehicle alone is administered by oral gavage,
and catalepsy was measured by the bar test every 30 min for
the next 2 h. Data were analyzed by one factor analysis of
variance with Dunnett’s t-test used to make post-hoc compari-
sons.
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Acknowledgment. We thank Azita Kaffashan for
carrying out the high-resolution MS. Ronald Savage and
Zhaoyang Li provided additional information regarding
rat pharmacokinetic data.
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available free of charge via the Internet at http://pubs.acs.org.
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