Synthesis and structure-activity relationship of pyrazolo[3,4- d]pyrimidines: Potent and selective adenosine A1 receptor antagonists

Article abstract of DOI:10.1021/jm960052s

A series of 12 substituted 1-phenylpyrazolo[3,4-d]pyrimidines were synthesized and evaluated for rat brain adenosine A₁ and A(2a) receptor binding affinity. Substituents at C-4 and C-6 were varied in order to define these regions in terms of molecular recognition by the receptor subtypes. At C-4, the effects of a mercapto, methylthio, and amino substituent were evaluated, while at C-6, amides with varying alky] groups extending from the α-carbon were examined. This study identified both potent and selective adenosine A₁ receptor antagonists. The most potent of the 12 compounds was α-[(4-amin-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl)thio]hexanamide (14); with an A₁ K(i) of 0.939 nM and an A(2a) K(i) of 88.3 nM, this compound is 94- fold A₁ selective. The most selective of the 12 compounds was α-[[4- (methylthio)-1-phenylpyrazolo[3,4-d]pyrimidin6-yl]thio]hexanamide (10); with an A₁ K(i) of 6.81 nM and an A(2a) K(i) > 40 000 nM, this compound is >5900- fold A, selective. The structure-activity relationships for the complete series has identified discrete structural differences between the A₁ and A(2a) receptors with respect to the binding of pyrazolo[3,4-d]pyrimidines. This study resulted in prediction that increased A₁ affinity could be achieved by incorporation of NH-alkyl substituents at C-4. This was confirmed by synthesis of α-[[4-(methylamino)-1-phenylpyrazolo[3,4-d]pyrimidin-6- yl]thio1]hexanamide (15) which was found to have an A₁ K(i) of 0.745 nM.

Full text of DOI:10.1021/jm960052s

4156
J . Med. Chem. 1996, 39, 4156-4161
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip of P yr a zolo[3,4-d ]p yr im id in es:
P oten t a n d Selective Ad en osin e A1 Recep tor An ta gon ists
Sally-Ann Poulsen and Ronald J . Quinn*
Queensland Pharmaceutical Research Institute, Griffith University, Brisbane 4111, Australia
Received J anuary 16, 1996X
A series of 12 substituted 1-phenylpyrazolo[3,4-d]pyrimidines were synthesized and evaluated
for rat brain adenosine A1 and A2a receptor binding affinity. Substituents at C-4 and C-6 were
varied in order to define these regions in terms of molecular recognition by the receptor subtypes.
At C-4, the effects of a mercapto, methylthio, and amino substituent were evaluated, while at
C-6, amides with varying alkyl groups extending from the R-carbon were examined. This study
identified both potent and selective adenosine A1 receptor antagonists. The most potent of
the 12 compounds was R-[(4-amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl)thio]hexanamide (14);
with an A1 Ki of 0.939 nM and an A2a Ki of 88.3 nM, this compound is 94-fold A1 selective. The
most selective of the 12 compounds was R-[[4-(methylthio)-1-phenylpyrazolo[3,4-d]pyrimidin-
6-yl]thio]hexanamide (10); with an A1 Ki of 6.81 nM and an A2a Ki > 40 000 nM, this compound
is >5900-fold A1 selective. The structure-activity relationships for the complete series has
identified discrete structural differences between the A1 and A2a receptors with respect to the
binding of pyrazolo[3,4-d]pyrimidines. This study resulted in prediction that increased A1
affinity could be achieved by incorporation of NH-alkyl substituents at C-4. This was confirmed
by synthesis of R-[[4-(methylamino)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thiol]hexanamide
(15) which was found to have an A1 Ki of 0.745 nM.
In tr od u ction
Adenosine is an endogenous nucleoside which medi-
ates a variety of important physiological responses by
interaction with specific adenosine receptors.1-3 These
receptors consist of seven transmembrane R-helices and
are G-protein coupled.3 There are four major adenosine
receptor subtypes which have been identified and cloned
from various mammalian tissue types: the A1, A2a, A2b,
and A3 subtypes.4 Despite the enormous therapeutic
F igu r e 1. 4,6-Bis[(R-carbamoylethyl)thio]-1-phenylpyrazolo-
[3,4-d]pyrimidine, the lead compound for this study.
potential for drugs interacting with these adenosine
receptor subtypes, new drugs which do so have not been
forthcoming.1-3,5 This situation is partly a consequence
of undesirable side effects due to the nonselectivity of
potential drug candidates for a unique adenosine recep-
tor subtype. Structure-activity studies leading to the
development of potent and subtype selective agonists
and antagonists of adenosine should allow delineation
of the specific requirements for binding to the individual
receptor subtypes. Such ligands would be valuable as
‘molecular tools’, probing the structural requirements
of the individual receptor subtype binding sites.
4,6-Bis[(R-carbamoylethyl)thio]-1-phenylpyrazolo[3,4-
d]pyrimidine (1) was identified as a novel adenosine A1
receptor antagonist, antagonizing adenosine-stimulated
cyclic adenosine monophosphate generation in guinea
pig brain slices.6,7 It has a Ki of 370 ( 60 nM at the A1
receptor and so is moderately potent. 1-Phenylpyrazolo-
[3,4-d]pyrimidine has been modified at C-4 with mer-
capto, methylthio, and amino substituents in order to
probe for hydrogen-bonding sites and steric tolerance.
At C-6, thioethers containing distal amides with varying
branched and linear alkyl groups extending from the
R-carbon were examined. This allowed exploration of
the steric and hydrophobic tolerance of the A1 and A2a
receptor subtypes. The structure-activity relationships
for these compounds have highlighted discrete differ-
ences in pyrazolo[3,4-d]pyrimidines with respect to the
A1 and A2a receptor binding, thus having implications
in the future design of more potent and more selective
ligands.
Ch em istr y
The synthetic route to the target pyrazolo[3,4-d]py-
rimidines is outlined in Scheme 1. Alkylation of 1-phen-
yl-5H,7H-pyrazolo[3,4-d]pyrimidine-4,6-dithione (2) at
the C-6 sulfur was achieved by stirring with a 1 mol
equiv of bromoalkylamide in pyridine at room temper-
ature (rt). The products (3-6) were collected by removal
of solvent and recrystallized from Me2SO and water. The
bromoalkylamides were prepared from the bromo acid
bromides or bromo acid chlorides by reaction with
ammonia. The site of alkylation of the monoalkylated
product, i.e., the sulfur at the C-4 or C-6 position, has
previously been determined using NMR spectroscopy
experiments to be at C-6.8
The C-4 methylthio series of compounds (7-10) were
synthesized by alkylation of the corresponding mercapto
series with iodomethane. A slight molar excess of
iodomethane and the required monoalkylated mercapto
compound were stirred in 1.5 M NaOH at room tem-
perature. The reaction products precipitated from solu-
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(96)00052-0 CCC: $12.00 © 1996 American Chemical Society

Synthesis and SAR of Pyrazolo[3,4-d]pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4157
Ta ble 1. Receptor Binding Results for
1-Phenyl-4,6-disubstituted pyrazolo[3,4-d]pyrimidines Binding
to Rat A1 and A2a Receptors
Sch em e 1. Synthetic Route to
1-Phenyl-4,6-disubstituted-pyrazolo[3,4-d]pyrimidinesa
A2a Ki (nM)b or A2a Ki/
compd
R4
SH
SH
SH
R6
Et
i-Pr 256 ( 4
A1 Ki (nM)a
% inhibtn (nM)c A1 Ki
3
4
155 ( 5
8750 ( 1100
44% (20 000)
4450 ( 790
56
g78
79
a
Reaction conditions: (i) BrCHRCONH2, pyridine; (ii) CH3I,
5
6
Pr
Bu
Et
56.3 ( 4.5
NaOH (aq); (iii) NH3 (g), EtOH, 110 °C or CH3NH2 (g), EtOH, 110
°C.
SH
37.2 ( 0.8
52% (10 000) g270
796 ( 135 95
37% (20 000) g1300
4380 ( 410 580
7
SCH3
SCH3
SCH3
SCH3
NH2
NH2
NH2
NH2
8.40 ( 0.32
8
9
i-Pr 15.7 ( 0.6
Pr
tion within 20 min, and the crude products were
recrystallized from Me2SO and water.
7.55 ( 2.32
6.81 ( 0.61
1.56 ( 0.09
2.73 ( 0.12
1.08 ( 0.30
0.939 ( 0.341
0.745 ( 0.045
10
11
12
13
14
15
Bu
Et
12% (40 000) >5900
Aminolysis of the methylthio compounds to give
compounds 11-14 was achieved by heating with etha-
nolic ammonia and to give compound 15 by heating with
ethanolic methylamine to 110 °C in a pressure-sealed
flask for 72 h. Workup of the reaction was achieved by
either the addition of ice cold water to precipitate the
products or removal of solvent. The crude products were
recrystallized from Me2SO and water.
Structural confirmation of target compounds was
achieved by NMR spectroscopy. 1H and 13C NMR
spectra were assigned with the aid of DEPT (distortion-
less enhancement by polarization transfer), HMQC
(heteronuclear multiple-quantum coherence), and HMBC
(heteronuclear multiple-bond coherence) NMR experi-
ments on a representative compound from each series.
44.5 ( 12.6
147 ( 17
35.3 ( 2.2
88.3 ( 3.8
247 ( 42
29
54
33
94
332
i-Pr
Pr
Bu
NHCH3 Bu
a
A1 receptor binding data utilizing competitive displacement
of specific [3H]-N6-PIA binding from A1 receptors in whole rat brain
membranes. Data are the average of at least two independent
experiments performed in duplicate and expressed as Ki ( SEM.
Kd of [3H]-N6-PIA was 1 nM. A2a receptor binding data utilizing
b
competitive displacement of specific [3H]CGS 21680 binding from
A2a receptors in rat striatal membranes. Data are the average of
at least two independent experiments performed in duplicate and
expressed as Ki ( SEM. Kd of [3H]CGS 21680 was 14.9 nM. c Lack
of solubility at high concentrations precluded determination of
IC50
.
chain the order was consistently R-butyl > R-propyl >
R-ethyl > R-isopropyl. The most potent compound was
14 which combined amino at C-4 with a R-butyl side
chain at C-6; it has an A1 Ki of 0.939 nM. The least
potent compound was 4, combining mercapto at C-4
with an R-isopropyl side chain at C-6, and it has an A1
Ki of 256 nM.
The structure-activity relationships exhibited by
these compounds at the A2a receptor are different from
those exhibited at the A1 receptor. At C-4 the order of
potency was amino > methylthio > mercapto (as it was
at the A1 receptor) where the C-6 side chain is R-ethyl,
R-isopropyl, and R-propyl. For the R-butyl side chain
this relationship did not hold, and the order of potency
was amino > mercapto > methylthio. The most potent
A2a compound was 13, with amino at C-4 and a R-propyl
side chain at C-6, and it has an A2a Ki of 35.3 nM. The
least potent A2a compound was 10, with methylthio at
C-4 and a R-butyl side chain at C-6, and it has a A2a Ki
of > 40 000 nM.
Ra d ioliga n d Bin d in g
The adenosine A1 and A2a receptor binding affinities
of the pyrazolo[3,4-d]pyrimidines were determined using
standard radioligand binding assay procedures.9,10 Com-
petitive binding assays against (R)-[3H]-N6-(phenyliso-
propyl)adenosine ([3H]-N6-PIA) using rat brain mem-
branes for A1 receptors and against [3H]-2-[[p-(2-car-
boxyethyl)phenethyl]amino]-5′-ethanecarboxamidoade-
nosine ([3H]CGS 21680) using rat striatal membranes
for A2a receptors were used to determine a full concen-
tration-inhibition curve for each compound. Analysis
of the results allowed calculation of an IC50 and corre-
sponding Ki value for each compound. These calcula-
tions used Kd values of 1 and 14.9 nM for [3H]-N6-PIA
and [3H]CGS 21680, respectively, in the Cheng-Prusoff
equation.11 The results for the receptor binding assays
are given in Table 1.
Resu lts
At both receptor subtypes the amino substituent at
C-4 is significantly more potent than the mercapto
substituent for all R-alkyl side chains at C-6. At the A1
receptor the increase in potency ranges from 40-fold for
the R-butyl side chain at C-6 to 99-fold for the R-ethyl
side chain at C-6. At the A2a receptor the increase in
potency is even more pronounced than at the A1 recep-
tor, increasing from 113-fold for the R-butyl side chain
at C-6 to 197-fold for the R-ethyl side chain at C-6.
The methylthio substituent at C-4 is more potent than
the mercapto substituent for all R-alkyl side chains at
C-6 at the A1 receptor and all but the R-butyl side chain
The structure-activity analysis of the series of 12
initial pyrazolo[3,4-d]pyrimidines 3-14 showed that the
C-4 and C-6 substituent effects were tightly linked. The
overall activity and subtype selectivity of each com-
pound reflected this interdependency and has enabled
us to identify discrete differences in the A1 and A2a
receptors in terms of binding of the pyrazolo[3,4-d]-
pyrimidines.
At the A1 receptor the effects of the C-4 and C-6
substituents were additive in terms of biological activity.
At C-4 the order of potency was consistently amino >
methylthio > mercapto, while for the C-6 R-alkyl side

4158 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Poulsen and Quinn
at the A2a receptor. At the A1 receptor the increase is
18.5-fold for R-ethyl, 16.3-fold for R-isopropyl, 7.5-fold
for R-propyl, and 5.5-fold for R-butyl side chains at C-6.
At the A2a receptor the improvement in potency is 11.0-
fold for the R-ethyl side chain; with the R-propyl and
R-isopropyl side chains the potency does not increase
significantly, while with the R-butyl side chain there is
a >4-fold drop in potency.
Discu ssion
Considering the C-4 position, the increase, of up to 2
orders of magnitude, in affinity at both receptor sub-
types of the amino substituent at C-4 compared to the
mercapto substituent at C-4 indicates a favorable
interaction between the amino substituent and the
receptor binding sites. As the ability to form hydrogen
bonds is the key difference between the amino and
mercapto substituents, the superiority of the amino
substituents potency most likely identifies a hydrogen-
bonding interaction with the receptor binding sites. The
structure-activity relationships between mercapto and
methylthio indicate the existence of a hydrophobic
pocket in both the A1 and A2a receptor binding sites
which accommodates the methyl group of the C-4
methylthio substituent. The favorable occupancy of this
pocket by the methyl group does however depend on the
nature of the alkyl group on the C-6 substituent.
Considering the C-6 position, the structure-activity
relationships exhibited by increasing the length of a
linear carbon chain extending from the R-carbon of the
amide substituent at C-6 provided information on steric
and hydrophobic tolerance of the A1 and A2a receptor
binding sites. The increase in length of the linear
carbon side chain from two to three to four carbons was
favorably tolerated at the A1 receptor for each C-4
substituent. At the A2a receptor the three-carbon R-pro-
pyl side chain was the favored group for mercapto and
amino at C-4, while increasing beyond this to the four-
carbon R-butyl side chain caused a reduction in potency
of 2.2- and 2.5-fold, respectively. For methylthio at C-4
the two-carbon R-ethyl was the favored side chain, as
the three-carbon R-propyl side chain led to a 5.5-fold
reduction in potency and the four-carbon R-butyl side
chain produced a further >9.1-fold reduction, in total a
>50.2-fold decrease.
Comparison of the R-ethyl and R-isopropyl side chains
showed that both the A1 and A2a receptor do not
favorably tolerate the increase in steric bulk introduced
by the â-methyl of the R-isopropyl substituent in place
of the â-hydrogen of the R-ethyl substituent. The
reduction in potency caused by introduction of the
â-branching ranges from 1.7- to 1.9-fold at the A1
receptor and from 2.3- to 25-fold at the A2a receptor.
The interdependency of the C-4 and C-6 substituent
effects in the structure-activity analysis of this series
of pyrazolo[3,4-d]pyrimidines is clearly evident as it is
a combination of the substituents that determines the
overall affinity of the ligands. Tolerance of the various
substituent changes is not the same for the A1 and A2a
receptor subtypes, and this influences the subtype
selectivity for each ligand. For the A1 receptor an amino
group is highly favored at C-4; however, there is also
room to favorably accommodate the methyl group of the
methylthio substituent, and hence it is likely that
improved potency can be achieved by incorporation of
F igu r e 2. Hypothetical C-4 and C-6 hydrophobic pockets of
adenosine A1 and A2a receptors. The combined C-4/C-6 hydro-
phobic pocket tolerance of alkyl carbons: (a) at the A1 receptor,
five, one at C-4 and four at C-6; at the A2a receptor, three, one
at C-4 and two at C-6 (for the methylthio substituent) or,
alternatively, (b) at the A2a receptor, three, zero at C-4 and
three at C-6 (for the mercapto or amino substituent).
NH-alkyl substituents at C-4. The size of the hydro-
phobic pocket at the R-carbon of the C-6 substituent is
sufficient to accommodate the four carbons of an R-butyl
side chain. At the A2a receptor the amino group is also
highly favored at C-4; again there is a small hydrophobic
pocket that can also accommodate the methyl of the
methylthio substituent. The alkyl carbon chain length
tolerated in the C-6 hydrophobic pocket is smaller than
at the A1 receptor. It can accommodate only three
carbons for the amino and mercapto groups at C-4 and
only two carbons for the C-4 methylthio series. The
hydrophobic pockets identified at C-4 and C-6 appear
not to be independent, and together they tolerate an
overall total alkyl carbon number, Figure 2. At the A1
receptor this is at least five carbons (one at C-4 and four
at C-6), and at the A2a receptor it is three carbons (zero
at C-4 and three at C-6 or one at C-4 and two at C-6).
The steric tolerance of the A2a receptor binding site
has been pushed to the outer limit by the variable C-6
R-alkyl side chains, and going beyond this limit leads
to A1 selective compounds. The least selective com-
pounds are those where the total alkyl carbon number
is three or less (compounds 3-5, 7, 8, and 11-13) as
these can bind favorably to either receptor subtype. The
compounds with four or five total alkyl carbons (com-
pounds 6, 9, 10, and 14) are the most A1 selective as
these bind favorably to the A1 receptor and unfavorably
to the A2a receptor, Figure 2. The most selective
compound was 10 with methylthio at C-4 and an R-butyl
side chain at C-6, and it has a total of five alkyl carbons
and is >5900-fold A1 selective, i.e., 4 orders of magni-
tude selective. The least selective compound is 11 with
amino at C-4 and an R-ethyl side chain at C-6, and it
has only two alkyl carbons (and so is tolerated by both
receptor subtypes) and is 29-fold A1 selective. The
amino substituent of 11 also contributes to this low
selectivity. This substituent is highly favored at both

Synthesis and SAR of Pyrazolo[3,4-d]pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4159
Ta ble 2. Chemical Data for Compounds 3-14
receptor subtypes but more so at the A2a receptor
(discussed above), and hence the A2a/A1 selectivity ratio
becomes even less. This is true for all the compounds
with amino at C-4: They exhibit lower selectivity than
the corresponding compounds with mercapto or meth-
ylthio at C-4.
The structure-activity relationships exhibited by
3-14 and the combined C-4/C-6 hydrophobic tolerance
proposed in Figure 2 suggested that 15, with methyl-
amino at C-4 and R-butyl side chain at C-6, would
exhibit improved A1 receptor affinity. The basis for
introduction of a methylamino substituent at C-4 in 15
was the identification of both a hydrogen-bonding
interaction and a small hydrophobic pocket within the
A1 receptor binding site. The combined C-4/C-6 hydro-
phobic pocket tolerance at the A1 receptor has been
proposed as five alkyl carbons (Figure 2), and the
R-butyl side chain at C-6 combined with the methyl-
amino at C-4 would conform to this requirement,
thereby providing an additional test for the proposal of
Figure 2. The corresponding compounds 10 and 14 with
an R-butyl side chain at C-6 were the most potent
compounds at the A1 receptor in the C-4 methylthio and
C-4 amino series, respectively. Compound 15 with a
methylamino at C-4 and an R-butyl side chain at C-6
was synthesized and evaluated.12 15 has an A1 Ki of
0.745 nM compared to an A1 Ki of 6.81 nM for the
corresponding C-4 methylthio compound 10 and an A1
Ki of 0.939 nM for the corresponding C-4 amino com-
pound 14. Consistent with Figure 2, 15 had decreased
A2a affinity and was 332-fold A1 selective. Both the
increased A1 affinity and the increased A1 selectivity
provide further evidence for the combined C-4/C-6
hydrophobic pocket tolerance (Figure 2).
yield
compd
R4
SH
SH
SH
SH
R6 (%)
mp (°C)
formula
anal.
3
4
5
6
7
8
9
Et
73 233-251 dec C15H15N5OS2 C, H, N, S
i-Pr 69 221-240 dec C16H17N5OS2 C, H, S; Na
Pr
Bu
91 222-232 dec C16H17N5OS2 C, H; Nb
80 221-241 dec C17H19N5OS2 C, H, S; Nc
SCH3 Et
86 223-224
C16H17N5OS2 C, H, N, S
C17H19N5OS2 C, H, N, S
C17H19N5OS2 C, H; Nd
C18H21N5OS2 C, H, N, S
SCH3 i-Pr 67 227-228
SCH3 Pr
69 222-224
59 219-221
10 SCH3 Bu
11 NH2
12 NH2
13 NH2
14 NH2
Et
69 250-258 dec C15H16N6OS C, H, S; Ne
i-Pr 70 240-254 dec C16H18N6OS C, H, S; Nf
Pr
Bu
70 235-244 dec C16H18N6OS C, H, N
85 234-247 dec C17H20N6OS C, H, N, S
15 NHCH3 Bu
74 198-199
C18H22N6OS C, H, S; Ng
N: calcd, 19.49; found, 18.9. b N: calcd, 19.49; found, 18.8. c N:
a
d
calcd, 18.75; found, 18.0. N: calcd, 18.75; found, 18.1. e N: calcd,
25.59; found, 25.0. f N: calcd, 24.55; found, 23.6. g N: calcd, 22.69;
found, 21.3.
refluxing over sodium and storage under nitrogen. Dry DMF
was prepared by stirring over barium oxide, under an atmo-
sphere of nitrogen for 24 h, followed by high vacuum distilla-
tion and storage over 4 Å molecular sieves. All other solvents
were analytical grade or distilled prior to use.
Melting points were determined on a Gallenkamp digital
melting point apparatus and are uncorrected. 1H, 13C, and
DEPT NMR spectra were recorded using a Bruker WM250 or
Varian Gemini-200 spectrometer. HMBC and HMQC experi-
ments were recorded with a Varian Unity-400 spectrometer.
IR spectra were recorded on a Perkin Elmer FTIR instrument,
with solid samples examined in KBr disks. Solvents were
removed under reduced pressure with a Buchi rotary evapora-
tor, DMF and Me2SO removal requiring high-vacuum ap-
paratus.
Microanalytical data were obtained from the Australian
Microanalytical Service. Compound purity was ascertained
by NMR, TLC, and electrospray mass spectrometry. High-
resolution mass spectra were obtained from the Organic Mass
Spectrometry Facility, University of Tasmania, Hobart, Tas-
mania, Australia.
1-Phenyl-5H,7H-pyrazolo[3,4-d]pyrimidine-4,6-dithione (2)
was prepared by known methods.8 A three-step procedure
involving synthesis of (ethoxymethylene)malononitrile and its
reaction with phenylhydrazine to give 5-amino-4-cyano-1-
phenylpyrazole followed by cyclization of this with potassium
O-ethylxanthogenate afforded 2.
r-[(4-Mer ca p t o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-
yl)th io]bu ta n a m id e (3). Meth od A: To a solution of 2
(0.500 g, 1.92 mmol) in dry pyridine (10 mL) was added 1 mol
equiv of 2-bromobutanamide (0.318 g, 1.92 mmol) in small
amounts over 20 min. The reaction mixture was stirred at
room temperature, and after 2 h a cream solid precipitated.
Solvent was removed from the reaction mixture under reduced
pressure; the cream solid that remained was collected by
suction filtration and washed with ice cold water. This crude
product was refluxed in ethanol and filtered while hot to
remove unreacted starting material, leaving a white solid.
Recrystallization of this solid from Me2SO and water afforded
Con clu sion s
This study has identified new compounds which are
highly potent (subnanomolar, e.g., 14 and 15) and highly
selective (4 orders of magnitude A1 selective, e.g., 10)
for adenosine A1 receptors over adenosine A2a receptors.
These new compounds were generated by the successful
optimization of the potency and selectivity exhibited by
the lead compound 1. The structure-activity relation-
ships for the complete series of compounds has high-
lighted clear differences with respect to the A1 and A2a
receptor binding of substituted pyrazolo[3,4-d]pyrim-
idines. In this context, the compounds of this study
have proved valuable in probing discrete structural
differences in the A1 and A2a receptor binding sites. In
particular, the differing steric tolerance of the hydro-
phobic pocket identified at C-6 and the hydrogen-
bonding requirements of the C-4 substituent have
potential for exploitation in the future design of more
potent and more selective pyrazolo[3,4-d]pyrimidines,
allowing a greater understanding of the structural
parameters of the A1 and A2a receptor subtype binding
sites.
Exp er im en ta l Section
1
All chemicals were obtained from Aldrich. Cylinders of
anhydrous ammonia were obtained from BOC gases. Dry
ethanol was prepared by refluxing distilled ethanol over
magnesium turnings and a catalytic quantity of iodine followed
by distillation and storage over 4 Å molecular sieves. Dry
pyridine was prepared by refluxing over sodium and storage
over potassium hydroxide. Dry hexane was prepared by
pure 3 as a white solid (yield 73%): mp 233-251 °C dec; H
NMR (200 MHz, Me2SO-d6) δ 0.97 (t, 3H, J ) 7.2 Hz, CH3),
1.95 (m, 2H, CH2), 4.32 (t, 1H, J ) 7.2 Hz, CH), 7.42 (br s, 1H,
NH), 7.43 (t, 1H, J ) 7.6 Hz, H-4′), 7.55 (dd, 2H, J ) 7.6 Hz,
H-3′, H-5′), 7.89 (br s, 1H, NH), 8.05 (d, 2H, J ) 7.6 Hz, H-2′,
H-6′), 8.34 (s, 1H, H-3), 14.10 (br s, 1H, SH); 13C NMR (50
MHz, Me2SO-d6) δ 11.6 (CH3), 25.8 (CH2), 50.8 (CH), 116.4

4160 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21
Poulsen and Quinn
(C-3a), 121.3 (C-2′, C-6′), 127.2 (C-4′), 129.4 (C-3′, C-5′), 138.0
(C-1′), 138.2 (C-3), 146.5 (C-7a), 160.3 (C-6), 171.0 (CdO), 180.2
(C-4); IR (KBr disk) 3393, 3289 (primary amide NH), 1657
(CdO), 1593 cm-1 (CdC).
(CH3), 30.5 (CH2), 55.8 (CH), 110.5 (C-3a), 120.8 (C-2′, C-6′),
126.7 (C-4′), 129.4 (C-3′, C-5′), 133.8 (C-3), 138.3 (C-1′), 150.9
(C-7a), 165.6 (C-4), 168.5 (C-6), 171.6 (CdO); IR (KBr disk)
3376, 3180 (primary amide NH), 1682 (CdO), 1596 cm-1
(CdC).
r-[(4-Mer ca p t o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-
yl)th io]-â-m eth ylbu ta n a m id e (4). Method A was used to
prepare 4 (yield 69%): mp 221-239 °C dec; 1H NMR (200
MHz, Me2SO-d6) δ 1.02 (d, 3H, J ) 6.8 Hz, CH3), 1.03 (d, 3H,
J ) 6.8 Hz, CH3), 2.33 (m, 1H, âCH), 4.38 (d, 1H, J ) 5.6 Hz,
CH), 7.38 (br s, 1H, NH), 7.40 (t, 1H, J ) 7.6 Hz, H-4′), 7.53
(dd, 2H, J ) 7.6 Hz, H-3′, H-5′), 7.80 (br s, 1H, NH), 8.07 (d,
2H, J ) 7.6 Hz, H-2′, H-6′), 8.34 (s, 1H, H-3), 14.10 (br s, 1H,
SH); 13C NMR (50 MHz, Me2SO-d6) δ 19.7 (CH3), 19.7 (CH3),
30.5 (âCH), 56.0 (CH), 116.4 (C-3a), 121.3 (C-2′, C-6′), 127.1
(C-4′), 129.4 (C-3′, C-5′), 138.0 (C-1′), 138.2 (C-3), 146.4 (C-
7a), 160.5 (C-6), 170.7 (CdO), 180.0 (C-4); IR (KBr disk) 3392,
3171 (primary amide NH); 1657 (CdO); 1573 cm-1 (CdC);
HRMS (chemical ionization, CI) 360.0952, calcd for C16H17N5-
OS2‚H+ 360.0953.
r-[(4-Mer ca p t o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-
yl)th io]p en ta n a m id e (5). Method A was used to prepare 5
(yield 91%): mp 222-232 °C dec; 1H NMR (200 MHz, Me2SO-
d6) δ 0.92 (t, 3H, J ) 7.2 Hz, CH3), 1.41 (m, 2H, γCH2), 1.94
(m, 2H, âCH2), 4.40 (t, 1H, J ) 7.2 Hz, CH), 7.37 (br s, 1H,
NH), 7.43 (t, 1H, J ) 7.6 Hz, H-4′), 7.53 (dd, 2H, J ) 7.6 Hz,
H-3′, H-5′), 7.87 (br s, 1H, NH), 8.09 (d, 2H, J ) 7.6 Hz, H-2′,
H-6′), 8.36 (s, 1H, H-3), 14.10 (br s, 1H, SH); 13C NMR (50
MHz, Me2SO-d6) δ 13.7 (CH3), 20.3 (γCH2), 34.6 (âCH2), 49.2
(CH), 116.4 (C-3a), 121.4 (C-2′, C-6′), 127.2 (C-4′), 129.3 (C-3′,
C-5′), 138.0 (C-1′), 138.2 (C-3), 146.4 (C-7a), 160.2 (C-6), 171.0
(CdO), 180.0 (C-4); IR (KBr disk) 3386, 3177 (primary amide
NH), 1657 (CdO), 1572 cm-1 (CdC); HRMS (CI) 360.0945,
calcd for C16H17N5OS2‚H+ 360.0953.
r-[[4-(Meth ylth io)-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -
6-yl]th io]p en ta n a m id e (9). Method B was used to prepare
1
9 (yield 69%): mp 222-224 °C; H NMR (200 MHz, Me2SO-
d6) δ 0.94 (t, 3H, J ) 7.2 Hz, CH3), 1.45 (m, 2H, γCH2), 1.93
(m, 2H, âCH2), 2.73 (s, 3H, SCH3), 4.45 (t, 1H, J ) 7.0 Hz,
CH), 7.25 (br s, 1H, NH), 7.42 (t, 1H, J ) 7.6 Hz, H-4′), 7.59
(dd, 2H, J ) 7.6 Hz, H-3′, H-5′), 7.78 (br s, 1H, NH), 8.18 (d,
2H, J ) 7.6 Hz, H-2′, H-6′), 8.53 (s, 1H, H-3); 13C NMR (50
MHz, Me2SO-d6) δ 11.7 (SCH3), 13.8 (CH3), 20.4 (γCH2), 34.7
(âCH2), 48.9 (CH), 110.5 (C-3a), 120.9 (C-2′, C-6′), 126.8 (C-
4′), 129.4 (C-3′, C-5′), 133.9 (C-3), 138.2 (C-1′), 151.0 (C-7a),
165.6 (C-4), 168.1 (C-6), 171.8 (CdO); IR (KBr disk) 3355, 3166
(primary amide NH), 1687 (CdO), 1593 cm-1 (CdC); HRMS
(electron impact, EI) 373.1040, calcd for C17H19N5OS2 373.1031.
r-[[4-(Meth ylth io)-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -
6-yl]th io]h exa n a m id e (10). Method B was used to prepare
10 (yield 59%): mp 219-221 °C; 1H NMR (200 MHz, Me2SO-
d6) δ 0.87 (t, 3H, J ) 6.9 Hz, CH3), 1.26 (m, 4H, δCH2γCH2),
1.93 (m, 2H, âCH2), 2.72 (s, 3H, SCH3), 4.43 (t, 1H, J ) 7.0
Hz, CH), 7.25 (br s, 1H, NH), 7.42 (t, 1H, J ) 7.6 Hz, H-4′),
7.59 (dd, 2H, J ) 7.6 Hz, H-3′, H-5′), 7.78 (br s, 1H, NH), 8.18
(d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.53 (s, 1H, H-3); 13C NMR
(62.8 MHz, Me2SO-d6) δ 11.5 (SCH3), 13.7 (CH3), 21.9 (δCH2),
29.2 (γCH2), 32.2 (âCH2), 49.0 (CH), 110.5 (C-3a), 120.9 (C-2′,
C-6′), 126.8 (C-4′), 129.4 (C-3′, C-5′), 133.9 (C-3), 138.3 (C-1′),
151.0 (C-7a), 165.8 (C-4), 168.2 (C-6), 172.0 (CdO); IR (KBr
disk) 3383, 3197 (primary amide NH), 1669 (CdO), 1619 cm-1
(CdC).
r-[(4-Mer ca p t o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-
yl)th io]h exa n a m id e (6). Method A was used to prepare 6
(yield 80%): mp 221-241 °C dec; 1H NMR (200 MHz, Me2SO-
d6) δ 0.85 (t, 3H, J ) 6.7 Hz, CH3), 1.33 (m, 4H, δCH2γCH2),
1.94 (m, 2H, âCH2), 4.40 (t, 1H, J ) 7.2 Hz, CH), 7.38 (br s,
1H, NH), 7.44 (t, 1H, J ) 7.6 Hz, H-4′), 7.57 (dd, 2H, J ) 7.6
Hz, H-3′, H-5′), 7.89 (br s, 1H, NH), 8.09 (d, 2H, J ) 7.6 Hz,
H-2′, H-6′), 8.37 (s, 1H, H-3), 14.10 (br s, 1H, SH); 13C NMR
(50 MHz, Me2SO-d6) δ 13.6 (CH3), 21.8 (δCH2), 29.0 (γCH2),
32.2 (âCH2), 49.3 (CH), 116.4 (C-3a), 121.4 (C-2′, C-6′), 127.2
(C-4′), 129.3 (C-3′, C-5′), 138.0 (C-1′), 138.2 (C-3), 146.5 (C-
7a), 160.3 (C-6), 171.2 (CdO), 181.0 (C-4); IR (KBr disk) 3381,
3164 (primary amide NH), 1658 (CdO), 1570 cm-1 (CdC);
HRMS (CI) 374.1106, calcd for C17H19N5OS2‚H+ 374.1109.
r-[[4-(Meth ylth io)-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -
6-yl]th io]bu ta n a m id e (7). Meth od B: Compound 3 (0.152
g, 0.44 mmol) was dissolved in 1.5 M NaOH (10 mL). A 1.5
mol excess of iodomethane (0.094 g, 0.66 mmol) was added and
the reaction mixture stirred at room temperature. After 10-
20 min a fine white precipitate had formed. The reaction
mixture was left stirring for 1 h. The precipitate was collected
by suction filtration and recrystallized from Me2SO and water,
producing fine white needles of pure 7 (yield 86%): mp 223-
r-[(4-Am in o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-yl)-
th io]bu ta n a m id e (11). Meth od C: Compound 7 (0.150 g,
0.46 mmol) was added to 20 mL of ethanolic ammonia,
prepared by saturating ethanol at 0 °C with ammonia gas. The
solution was placed in a bomb, sealed, and heated in an oil
bath preheated at 110 °C for 72 h. The reaction product
precipitated on cooling; ice cold water was added to the bomb
to aid any further precipitation of product. The crude product
was collected by suction filtration and recrystallized from
ethanol and water (yield 69%): mp 250-258 °C dec; 1H NMR
(200 MHz, Me2SO-d6) δ 1.01 (t, 3H, J ) 7.2 Hz, CH3), 1.94 (m,
2H, CH2), 4.26 (t, 1H, J ) 7.0 Hz, CH), 7.19 (br s, 1H, NH),
7.35 (t, 1H, J ) 7.6 Hz, H-4′), 7.55 (dd, 2H, J ) 7.6 Hz, H-3′,
H-5′), 7.68 (br s, 1H, NH), 7.95 (br s, 1H, NHamine), 8.06 (br
s, 1H, NHamine), 8.22 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.28 (s,
1H, H-3); 13C NMR (62.8 MHz, Me2SO-d6) δ 11.8 (CH3), 25.7
(CH2), 49.5 (CH), 99.4 (C-3a), 120.4 (C-2′, C-6′), 126.0 (C-4′),
129.2 (C-3′, C-5′), 134.4 (C-3), 139.0 (C-1′), 153.6 (C-7a), 157.5
(C-4), 169.0 (C-6), 172.3 (CdO); IR (KBr disk) 3490 (NH); 3379,
3171 (primary amide NH), 1652 (CdO), 1588 cm-1 (CdC);
HRMS (EI) 328.1106, calcd for C15H16N6OS 328.1106.
r-[(4-Am in o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-yl)-
th io]-â-m eth ylbu ta n a m id e (12). Method C was used to
prepare 12 (yield 70%): mp 240-254 °C dec; 1H NMR (250
MHz, Me2SO-d6) δ 1.03 (d, 6H, J ) 6.5 Hz, 2 × CH3), 2.23 (m,
1H, âCH), 4.27 (d, 1H, J ) 6.7 Hz, CH), 7.15 (br s, 1H, NH),
7.32 (t, 1H, J ) 7.6 Hz, H-4′), 7.52 (dd, 2H, J ) 7.6 Hz, H-3′,
H-5′), 7.56 (br s, 1H, NH), 7.92 (br s, 1H, NHamine), 8.08 (br
s, 1H, NHamine), 8.20 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.25 (s,
1H, H-3); 13C NMR (62.8 MHz, Me2SO-d6) δ 19.8 (CH3), 20.0
(CH3), 30.2 (CH), 54.8 (CH), 99.4 (C-3a), 120.4 (C-2′, C-6′),
126.0 (C-4′), 129.2 (C-3′, C-5′), 134.3 (C-3), 139.0 (C-1′), 153.6
(C-7a), 157.4 (C-4), 169.2 (C-6), 172.2 (CdO); IR (KBr disk)
3494 (NH), 3383, 3167 (primary amide NH), 1669 (CdO), 1594
cm-1 (CdC); HRMS (EI) 342.1276, calcd for H18N6OS 342.1263.
r-[(4-Am in o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-yl)-
th io]p en ta n a m id e (13). Method C was used to prepare 13
(yield 70%): mp 235-244 °C dec; 1H NMR (200 MHz, Me2SO-
d6) δ 0.94 (t, 3H, J ) 7.2 Hz, CH3), 1.42 (m, 2H, γCH2), 1.87
(m, 2H, âCH2), 4.33 (t, 1H, J ) 7.2 Hz, CH), 7.16 (br s, 1H,
NH), 7.35 (t, 1H, J ) 7.6 Hz, H-4′), 7.54 (dd, 2H, J ) 7.6 Hz,
H-3′, H-5′), 7.65 (br s, 1H, NH), 7.93 (br s, 1H, NHamine), 8.02
(br s, 1H, NHamine), 8.21 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.29
1
224 °C; H NMR (200 MHz, Me2SO-d6) δ 1.00 (t, 3H, J ) 7.2
Hz, CH3), 1.95 (m, 2H, CH2), 2.68 (s, 3H, SCH3), 4.36 (t, 1H, J
) 6.9 Hz, CH), 7.26 (br s, 1H, NH), 7.38 (t, 1H, J ) 7.6 Hz,
H-4′), 7.56 (dd, 2H, J ) 7.6 Hz, H-3′, H-5′), 7.79 (br s, 1H,
NH), 8.14 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.49 (s, 1H, H-3); 13
C
NMR (50 MHz, Me2SO-d6) δ 11.7 (SCH3 or CH3), 11.8 (SCH3
or CH3), 25.9 (CH2), 50.5 (CH), 110.5 (C-3a), 120.9 (C-2′, C-6′),
126.8 (C-4′), 129.4 (C-3′, C-5′), 133.9 (C-3), 138.2 (C-1′), 150.9
(C-7a), 165.6 (C-4), 168.1 (C-6), 171.7 (CdO); IR (KBr disk)
3346, 3166 (primary amide NH), 1687 (CdO), 1593 cm-1
(CdC).
â-Met h yl-r-[[4-(m et h ylt h io)-1-p h en ylp yr a zolo[3,4-d ]-
p yr im id in -6-yl]th io]bu ta n a m id e (8). Method B was used
to prepare 8 (yield 67%): mp 227-228 °C; 1H NMR (250 MHz,
Me2SO-d6) δ 1.04 (d, 3H, J ) 7.0 Hz, CH3), 1.07 (t, 3H, J ) 7.0
Hz, CH3), 2.31 (m, 1H, âCH), 2.70 (s, 3H, SCH3), 4.41 (d, 1H,
J ) 5.8 Hz, CH), 7.25 (br s, 1H, NH), 7.38 (t, 1H, J ) 7.6 Hz,
H-4′), 7.57 (dd, 2H, J ) 7.6 Hz, H-3′, H-5′), 7.70 (br s, 1H,
NH), 8.16 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.48 (s, 1H, H-3); 13
C
NMR (62.8 MHz, Me2SO-d6) δ 11.6 (SCH3), 19.6 (CH3), 20.1

Synthesis and SAR of Pyrazolo[3,4-d]pyrimidines
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 21 4161
(s, 1H, H-3); 13C NMR (50 MHz, Me2SO-d6) δ 13.8 (CH3), 20.5
(âCH2), 34.8 (γCH2), 47.9 (CH), 99.4 (C-3a), 120.4 (C-2′, C-6′),
126.1 (C-4′), 129.1 (C-3′, C-5′), 134.4 (C-3), 139.0 (C-1′), 153.6
(C-7a), 157.4 (C-4), 168.9 (C-6), 172.4 (CdO); IR (KBr disk)
3492 (NH), 3388, 3180 (primary amide NH), 1651 (CdO), 1586
cm-1 (CdC).
r-[(4-Am in o-1-p h en ylp yr a zolo[3,4-d ]p yr im id in -6-yl)-
th io]h exa n a m id e (14). Method C was used to prepare 14
(yield 85%): mp 234-247 °C dec; 1H NMR (250 MHz, Me2SO-
d6) δ 0.84 (t, 3H, J ) 6.7 Hz, CH3), 1.31 (m, 4H, δCH2γCH2),
1.86 (m, 2H, âCH2), 4.29 (t, 1H, J ) 7.1 Hz, CH), 7.16 (br s,
1H, NH), 7.33 (t, 1H, J ) 7.6 Hz, H-4′), 7.51 (dd, 2H, J ) 7.6
Hz, H-3′, H-5′), 7.67 (br s, 1H, NH), 7.92 (br s, 1H, NHamine),
8.07 (br s, 1H, NHamine), 8.18 (d, 2H, J ) 7.6 Hz, H-2′, H-6′),
8.26 (s, 1H, H-3); 13C NMR (62.8 MHz, Me2SO-d6) δ 13.8 (CH3),
21.9 (âCH2), 29.3 (γCH2), 32.3 (δCH2), 48.0 (CH), 99.4 (C-3a),
120.5 (C-2′, C-6′), 126.1 (C-4′), 129.2 (C-3′, C-5′), 134.4 (C-3),
139.0 (C-1′), 153.7 (C-7a), 157.6 (C-4), 169.0 (C-6), 172.5 (CdO);
IR (KBr disk) 3464 (NH), 3352, 3158 (primary amide NH),
1668 (CdO), 1587 cm-1 (CdC).
r-[[(4-(Meth yla m in o)-1-p h en ylp yr a zolo[3,4-d ]p yr im i-
d in -6-yl]th io]h exa n a m id e (15). R-[[4-(Methylthio)-1-phe-
nylpyrazolo[3,4-d]pyrimidin-6-yl]thio]hexanamide (10) (0.190
g, 0.490 mmol) was added to ethanolic methylamine (15 mL,
prepared by saturating ethanol at 0 °C with methylamine gas).
The solution was placed in a bomb, sealed, and heated in an
oil bath at 110 °C. After 72 h the bomb was cooled to 0 °C.
The product precipitated on cooling, ice cold water was added,
and the crude product was collected and recrystallized from
Me2SO and water affording pure 15 as a white solid (yield
74%): mp 198.3-199.3 °C dec; 1H NMR (200 MHz, Me2SO-
d6) δ 0.87 (t, 3H, J ) 7.2 Hz, CH3), 1.37 (m, 4H, δCH2, γCH2),
1.90 (m, 2H, âCH2), 3.00 (d, 3H, J ) 4.6 Hz, NHCH3), 4.36 (t,
1H, J ) 7.2 Hz, CH), 7.16 (br s, 1H, NH), 7.35 (t, 1H, J ) 7.6
Hz, H-4′), 7.54 (dd, 2H, J ) 7.6, 7.6 Hz, H-3′, H-5′), 7.68 (br s,
1H, NH), 8.20 (d, 2H, J ) 7.6 Hz, H-2′, H-6′), 8.27 (s, 1H, H-3),
8.54 (q, 1H, J ) 4.6 Hz, NHCH3); 13C NMR (50 MHz, Me2SO-
d6) δ 14.0 (CH3), 22.1 (δCH2), 27.0 (NCH3), 29.5 (γCH2), 32.6
(âCH2), 48.5 (CH), 99.8 (C-3a), 120.4 (C-2′, C-6′), 126.1 (C-4′),
129.1 (C-3′, C-5′), 133.8 (C-3), 138.9 (C-1′), 153.0 (C-7a), 156.0
(C-4), 168.9 (C-6), 172.4 (CdO); IR (KBr disk) 3300 (NH), 3379,
3180 (primary amide NH), 1670 (CdO), 1598 cm-1 (CdC);
HRMS (EI) 370.1567, calcd for C18H22N6OS 370.1576. Anal.
Calcd (C18H22N6OS): C, 58.35; H, 6.00; N, 22.69; S, 8.65.
Found: C, 58.7; H, 6.0; N, 21.3; S, 8.8.
Ad en osin e A1 Recep tor Bin d in g Assa y. Compounds
were assessed for their ability to inhibit binding of the A1
selective agonist radioligand [3H]-N6-PIA to membranes from
rat whole brain using a literature procedure modified for
automation. Receptor binding assays were carried out in 96-
well microtiter plates in an assay volume of 200 µL. Each
assay contained membrane (100 µg, pretreated with 0.1 U/mg
adenosine deaminase for 10 min at 37 °C), 1 nM (R)-[3H]-N6-
PIA (Amersham; 61 Ci/mmol), incubation buffer (50 mM Tris‚-
HCl, pH 7.4), and test compound (at least 12 concentrations)
in Me2SO giving a final Me2SO concentration of 1%, 2%, or
5%. Both 1% and 2% Me2SO did not decrease control binding,
while 5% Me2SO decreased control binding by 17%. Nonspe-
cific binding was determined in the presence of 10 µM
2-chloroadenosine. The assay was incubated for 90 min at 25
°C and then filtered using a cell harvester (Tomtec Harvester
96) onto untreated glass fiber B filtermats. All assays were
performed a minimum of two times with duplicate determina-
tions. Known compounds were used to characterize this assay
procedure. Data were fitted to optimized models by nonlinear
regression analysis (Graphpad Inplot IV, San Diego, CA).
Analysis of the results allowed calculation of an IC50 and
corresponding Ki value for each compound using a Kd value of
1 nM for [3H]-N6-PIA and the Cheng-Prusoff equation.
Ad en osin e A2a Recep tor Bin d in g Assa y. Compounds
were assessed for their ability to inhibit the binding of the A2a
agonist radioligand [3H]CGS 21680 to rat striatal membranes.
The assay volume was 250 µL. Each assay contained striatal
membrane (150 µg, pretreated as above), 5 nM [3H]CGS 21680
(New England Nuclear; 48.6 Ci/mmol), incubation buffer (50
mM Tris‚HCl, 10 mM MgCl2, pH 7.4), and test compound (at
least 12 concentrations) in Me2SO giving a final Me2SO
concentration of 1%, 2%, or 5%. Both 1% and 2% Me2SO
decreased control binding by 9%, while 5% Me2SO decreased
control binding by 24%. Nonspecific binding was determined
in the presence of 20 µM 2-chloroadenosine. The assay was
incubated for 120 min at 25 °C. Subsequent harvesting of the
assay was identical with the A1 assay. Results from concen-
tration-response curves were analyzed with Graphpad Inplot
IV, as above. Analysis of the results allowed calculation of
an IC50 and corresponding Ki value for each compound using
a Kd value of 14.9 nM for [3H]CGS 21680 and the Cheng-
Prusoff equation.
Ack n ow led gm en t. Support of this work by the
National Health and Medical Research Council and the
bequest of the late Thomas Patrick Hinch is gratefully
acknowledged. We acknowledge the award of an Aus-
tralian Postgraduate Award to S.P. We thank Roger
Moni for advice on radioligand binding assays.
Refer en ces
(1) Mu¨ller, C. E.; Scior, T. Adenosine receptors and their modulators.
Pharm. Acta Helv. 1993, 68, 77-111.
(2) Collis, M. G.; Hourani, S. M. O. Adenosine receptor subtypes.
TIPS 1993, 14, 360-366.
(3) J acobson, K. A.; van Galen, P. J . M.; Williams, M. Adenosine
receptors: pharmacology, structure-activity relationships, and
therapeutic potential. J . Med. Chem. 1992, 35, 407-422.
(4) J acobson, M. Cloning and expression of human adenosine
receptor subtypes. In Adenosine and Adenine Nucleotides: From
Molecular Biology to Integrative Physiology; Bellardinelli, L.,
Pelleg, A., Eds.; Kluwer: Norwell, MA, 1995; pp 5-13.
(5) Erfurth, A.; Schmauss, M. Treating neuropsychiatric disorders
with adenosinergic drugs-new perspective. Fortschr. Neurol.,
Psychiatr. 1995, 63, 93-98.
(6) Davies, L. P.; Brown, D. J .; Chow, S. C.; J ohnston, G. A. R.
Pyrazolo [3,4-d]]pyrimidines, a new class of adenosine antago-
nists. Neurosci. Lett. 1983, 41, 189-193.
(7) Davies, L. P.; Chow, S. C.; Skerritt, J . H.; Brown, D. J .; J ohnston,
G. A. R. Pyrazolo[3,4-d]pyrimidines as adenosine antagonists.
Life Sci. 1984, 34, 2117-2128.
(8) Quinn, R. J .; Scammells, P. J .; Tucker, D. J . Mono-R-carbam-
oylethylthio-substituted pyrazolo[3,4-d]pyrimidines: the position
of substitution. Aust. J . Chem. 1991, 44, 753-757.
(9) Schwabe, U.; Trost, T. Characterization of adenosine receptors
in rat brain by (-)[3H]N6-phenylisopropyladenosine. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1980, 313, 179-188.
(10) J arvis, M. F.; Schulz, R.; Hutchison, A. J .; Do, U. H.; Sills, M.
A.; Williams, M. [3H]CGS21680, a selective A2 adenosine receptor
agonist directly labels A2 receptors in rat brain. J . Pharmacol.
Exp. Ther. 1989, 251, 888-893.
(11) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibition which causes
50 percent inhibition (IC50) of enzyme reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(12) Poulsen, S. A.; Quinn, R. J . Pyrazolo[3,4-d]pyrimides: C4, C6
Substitution leads to Adenosine A1 receptor selectivity. Bioorg.
Med. Chem. Lett. 1996, 6, 357-360.
J M960052S