1-Phenylpyrazolo[3,4-d]pyrimidines; structure-activity relationships for C6 substituents at A1 and A(2A) adenosine receptors

Article abstract of DOI:10.1016/S0968-0896(00)00190-5

Substitution of 1-phenylpyrazolo[3,4-d]pyrimidines at C6 with N-alkyl-2-thiopropionamide groups has resulted in a series of 18 compounds which have been evaluated for binding at A₁ and A(2A) adenosine receptors. Introduction of an N-ethyl group gave increased affinity at both A₁ and A(2A) receptors for the amino compound 7b compared to the primary amide 7a. An additional hydrophobic pocket exists for substituents on the amide. This pocket allows an N-ethyl group for increased affinity at both A₁ and A(2A) receptors, allows larger alkyl groups at A(2A) receptors but not at A₁ receptors and there is an H-bond interaction requiring one H-bond donor. Molecular modeling studies have also enabled a proposal of the amino acid residues involved in ligand binding at both the A₁ and A(2A) receptors. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00190-5

Bioorganic & Medicinal Chemistry 8 (2000) 2581±2590
1-Phenylpyrazolo[3,4-d ]pyrimidines; Structure±Activity
Relationships for C6 Substituents at A1 and A2A
Adenosine Receptors
Mary Chebib, Declan McKeveney and Ronald J. Quinn*
AstraZeneca R&D Grith University, Brisbane 4111, Australia
Received 6 January 2000; accepted 5 July 2000
AbstractÐSubstitution of 1-phenylpyrazolo[3,4-d]pyrimidines at C6 with N-alkyl-2-thiopropionamide groups has resulted in a ser-
ies of 18 compounds which have been evaluated for binding at A1 and A2A adenosine receptors. Introduction of an N-ethyl group
gave increased anity at both A1 and A2A receptors for the amino compound 7b compared to the primary amide 7a. An additional
hydrophobic pocket exists for substituents on the amide. This pocket allows an N-ethyl group for increased anity at both A1 and
A2A receptors, allows larger alkyl groups at A2A receptors but not at A1 receptors and there is an H-bond interaction requiring one
H-bond donor. Molecular modeling studies have also enabled a proposal of the amino acid residues involved in ligand binding at
both the A1 and A2A receptors. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
activity with renal vasodilatory and uricosic e€ects. In
addition, FK 453 markedly antagonized the negative
inotropic e€ects of adenosine in isolated guinea-pig
atria but was less potent in inhibiting the relaxation
induced by adenosine in guinea pig aorta. These results
suggested that FK 453 is a potent and selective adenosine
A1 antagonist that could be useful in the treatment of
hypertension and renal failure. FK 453 produced dose-
dependent diuretic, natriuretic and uricosuric e€ects in
healthy human subjects and in patients with essential
hypertension.7
Adenosine and its analogues exert physiological
responses through four major subtypes of adenosine
receptors, A1, A2A, A2B, and A3. The responses act via
G-coupled proteins consisting of seven transmembrane
helices which activate several e€ector systems including
adenylate cyclase, potassium and calcium channels,
phospholipase A2 or C and guanylate cyclase.1 3
The ®rst group of AR antagonists that will enter the
market appears as if it will be the A1-selective antago-
nists.4 Compounds like KFM19 and MDL 102503 are
targeted for CNS disorders such as Morbus Alzheimer
and as general cognition enhancers in geriatric therapy.
A1-selective antagonists, such as KW3902 and FK 453,
are currently developed as potassium-saving diuretics
with kidney-protective properties, and for the treatment
of acute renal failure.4 Structural requirements for
selective antagonists for the A1 and A2A receptors have
been reviewed.5,6 FK 453 is a novel non-xanthine
antagonist that is 657-fold selective for binding to A1
versus A2A receptors and is under development for the
treatment of hypertension and renal failure.6 FK 453,
(+)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)acryl-
oyl]-2-piperidine ethanol, is a novel pyrazolopyridine
derivative that was found to possess a potent diuretic
Pyrazolo[3,4-d]pyrimidines were originally identi®ed as
adenosine A1 antagonists during a study of a large
number of nitrogen heterocycles related to ca€eine and
theophylline. The most active compound of the study
was 4,6-bis-a-carbamoylethylthio-1-phenylpyrazolo[3,4-d]
pyrimidine (1) with a Ki of 370Æ60 nM at the A1 recep-
tor (Fig. 1).8,9 We have recently reported the synthesis
and receptor binding at A1 and A2A receptors of a series
of 1-phenylpyrazolo[3,4-d]pyrimidines substituted at C6
with thioethers containing distal amide substituents and
substituted at C4 with thiol, thiomethyl and amino
substituents. The most selective 1-phenylpyrazolo[3,4-d]
pyrimidine for the A1 receptor was 20-(4-amino-1phenyl-
pyrazolo[3,4-d]pyrimidin-6-ylthio)N-ethyl-ethanamide
(2) with a KiA1 of 12.1Æ4.5 nM and a KiA2A of 131Æ36
nM and is a modest 10.8 times more selective for this
receptor.10,11 The most selective 1-phenylpyrazolo [3,4-d]
pyrimidine for the A2A was 30-(4-amino-1-phenylpyrazolo
*Corresponding author. Tel.: +61-7-3875-6006; fax: +61-7-3875-
6001; e-mail: r.quinn@az.gu.edu.au
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00190-5

2582
M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
[3,4-d]pyrimidin-6-ylthio)propanamide (3) with a KiA1
of 428Æ25 nM and a KiA2A of 101Æ26 nM and is a
modest 4.2 times more selective for this receptor. This
was gained mainly by decreased A1 anity while the A2A
anity remained relatively una€ected. Increasing the
methylene bridge by one carbon resulted in a decrease in
A1 receptor anity. This change was tolerated by the A2A
receptor. Increasing the methylene bridge further resul-
ted in a decrease in both A1 and A2A receptor ani-
ties.11 The study also showed that compounds
substituted with an amino group in the C4 position had
higher anity for both A1 and A2A receptors than both
the thiol and thiomethyl substituents. The thiomethyl
compounds had greater anity than the thiol com-
pounds. It was concluded that for high anity at both
A1 and A2A adenosine receptors the distal amide should
be separated from the C6 thiol by only one carbon.
further and re®ned to leave us with the binding site
models proposed here. Both receptor models were in
close agreement with previous binding study results.17
All molecular modeling was carried out using the
We now report the e€ect of varying the amide group
and keeping the distance between the amide and the C6
thiol at one carbon. These compounds contain a thiol,
thiomethyl, thioalkylamide and an amino group in the
C4 position.
Figure 1.
Results
1-Phenyl-5H,7H-pyrazolo[3,4-d]pyrimidine dithione (4)
was monoalkylated as previously described12 with the
corresponding bromoamides to yield compounds 5(b±e),
alkylated with methyl iodide to yield compounds 6(b±f)
and ®nally converted to the 4-amino compounds, com-
pounds 7(b±f), by treatment with ethanolic ammonia in
a sealed tube (Scheme 1). The bisalkylated compounds
8(b±f) were synthesized using 2 molar equivalents of the
bromoamides in aqueous sodium hydroxide and t-but-
anol (Scheme 2). Receptor bindings at rat membrane
adenosine A1 and A2A receptors are given in Table 1.
Molecular Modeling
The models for both the human A1 and the A2A ade-
nosine receptors were based on structures imported
from the G-Protein Coupled Receptor Database
(GPCRD).13 These consisted of the seven transmem-
brane helices. The helical bundles were then minimized,
with tethering, using the Powell method with Kollman
force ®eld and charges. Gradient was used as the termi-
nation criteria.
Scheme 1.
The loops (including amino- and carboxy-terminus
strands) were then constructed and attached to the
minimized helices. The antagonist structures modeled
were then constructed and, after an initial clean-up, mini-
mized using the conjugate gradient method. Docking
experiments were then carried out using the ¯exidock
program based on a variety of parameters such as charge
and distance constraints within the general region of the
receptor indicated by mutagenesis studies14,15 and
molecular modeling16 to be important in binding. Many
ligands were used for docking to explore possible bind-
ing sites and the most `sensible' solutions were examined
Scheme 2.

M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
Table 1. Receptor binding at rat membrane adenosine A1 and A2A receptors
2583
Compound
R1
R2
R3
A1 Receptor
Ki, nM
%
Inhibition
A2A Receptor
Ki, nM
%
Inhibition
KiA2A/KiA1
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
H
H
H
Et
H
H
H
H
H
Et
Ð
Ð
Ð
Ð
Ð
H
Et
Pr
Bu
SH
SH
SH
SH
587Æ91
240Æ22
402Æ64
0.68
10.5
2520Æ450
0
5
0
5
6
17
Cyclopentyl
SH
H
Et
Pr
Bu
SMe
SMe
SMe
SMe
SMe
SMe
NH2
NH2
NH2
NH2
NH2
NH2
Ð
30.3Æ2.9
60.8Æ8.2
39.5Æ6.4
272Æ33
1.30
4.47
39
26
21
2
34
2
10
3
Cyclopentyl
Et
H
Et
Pr
Bu
17.4Æ1.6
7.13Æ1.32
72.4Æ15.2
329Æ67
18.5Æ2.3
10.0Æ2.2
185Æ29
196Æ31
1.06
1.40
2.56
0.59
Cyclopentyl
Et
34
22
890Æ170
229Æ20
364Æ64
1420Æ425
146Æ27
1.60
0.64
2.39
8a
8b
8c
8d
8e
H
Et
Pr
Ð
Ð
Ð
Ð
425Æ49
0
2
0
0
3
0
Bu
Cyclopentyl
SYBYL 6.6 (Tripos Inc, St Louis, MO, USA) software
package which was run on a Silicon Graphics Octane
workstation.
there is a tolerance for larger N-alkyl substituents at the
C6 position for the A2A receptor than the A1 receptor.
With N,N-dialkyl substituents, there was a loss in a-
nity for both the A1 and A2A receptors, suggesting pos-
sible hydrogen bonding of the amide group to the
receptor. Compound 3 still remains to be the most
selective compound for the A2A receptor while com-
pound 7d is the most selective compound of this series.
Although we do not observe a more selective compound
for the A2A receptor, it may be worth exploring N-alkyl
substituted compounds using compound 3 as the parent
structure for further optimization and development in
A2A selective antagonists.
Discussion
The synthetic compounds were compared to the pre-
viously synthesized primary amides 5a, 6a, 7a, 8a.12
The trends observed with this extended group of com-
pounds were similar to the trends observed pre-
viously.11 The thiol compounds 5(a±e), and the
bisalkylated compounds 8(a±e), had lower anity for
the A1 and A2A receptors than the thiomethyl com-
pounds 6(a±f) (Table 1). The thiomethyl compounds
had lower anity for the A1 and A2A receptors than the
amino compounds 7(a±f). Introduction of the N-ethyl
group gave increased anity at both A1 and A2A
receptors for the amino compound 7b compared to 7a.
As the N-alkyl group increased from an ethyl, com-
pound 7b, to a propyl group, compound 7c, the A1 and
the A2A anity decreased. The A1 anity was further
decreased with the butyl compounds 7d, but the A2A
anity remained relatively unchanged. There is a 46-
fold loss in anity of compound 7d at the A1 receptor
when compared to compound 7b, and a 19.6-fold loss in
anity at the A2A receptor. This results in a 2.4-fold
increase in A2A receptor selectivity. This suggests that
Conclusion
At both the A1 and A2A receptors, the N-ethyl com-
pounds have the highest anity followed by the primary
amides. As the alkyl chain increases, anity at both
receptors decreases. This study allows a further de®ni-
tion of the previously published model17 as shown in
Figure 2. An additional hydrophobic pocket exists for
substituents on the amide. This pocket allows an
N-ethyl group for increased anity at both A1 and A2A
receptors, allows larger alkyl groups at A2A receptors
but not at A1 receptors and there is an H-bond interac-
tion requiring one H-bond donor.

2584
M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
Figure 2. Diagrams (a) and (b) represent previously published work:17 (a) the A1 receptor with summary of hydrophobic pocket carbon tolerance and the
2A boundary with a methylthio group at C4; (b) it was postulated that the A2A receptor C6 hydrophobic pocket could tolerate either 2 or 3 carbons
A
depending on whether a methylthio substituent is present at C4 or not. Diagrams (c) and (d) represent binding data and modeling results proposed by this
study; (c) identi®cation of amino acids representing the binding site for the A1 receptor. The N-alkyl pocket has a limit of 2 carbons; (d) the binding site
model for the A2A receptor proposes a tolerance for larger substituents at the N-alkyl pocket. Interactions of various amino acids with the ligand are
indicated with arrows. The transmembrane helix on which the amino acids are found is also shown. Dashed lines represent H-bonding.
As shown in Fig. 2, the proposed binding site from
molecular modeling studies allowed amino acid residues
potentially involved in ligand binding to be identi®ed.
into a hydrophobic area de®ned by residues Ile-19, Ala-
20, Leu-21 and Val-22, all on helix I. This allows enough
space to tolerate 4 alkyl carbons. The C6 alkyl chain at
the A2A receptor can extend into a smaller hydrophobic
pocket de®ned by residues Ala-63, Ile-64, Leu-272 and
Ala-273. The pocket is therefore formed between helices
II and VII and can tolerate only 3 alkyl carbons.
At the A1 receptor an H-bond is observed between the
carbonyl oxygen of Leu-90 and the C4 amino sub-
stituent. The A2A receptor shows similar H-bonding but
this time to Thr-88. This interaction would explain the
preference for an amine over a thiol or thiomethyl sub-
stituent at C4. It was also observed that in both receptors
an alkyl region or `roof' was present far enough above
the H-bonding residue to allow a thiomethyl group to
interact, thereby o€ering a possible explanation for the
preference of thiomethyl over thiol at both receptors.
The residues Val-87 and Leu-88, making up this region
for the A1 receptor, have previously been postulated to
have a role in ligand binding.18
At the A1 receptor the C6 amide hydrogen was seen to
be capable of acting as an H-bond donor to either Val-
58 and Gly-59 as was predicted from the binding a-
nities. There was no H-bonding observed for the amide
at the A2A receptor, which could go some way to
explaining the selectivity shown for the A1 receptor.
The N-alkyl pockets investigated in this study were
observed in both binding site models. For the A1 recep-
tor, the pocket is formed by the coiling of hydrophobic
residues Val-58, Gly-59, Ala-60 and Leu-61, all of which
are on helix II. This tight coiling around the alkyl chain
will permit two alkyl carbons only. The corresponding
hydrophobic pocket on the A2A receptor is formed by
The limitations of the C6 hydrophobic pocket had been
explored in previous binding studies17 and the receptor±
ligand models were in agreement with these results. For
the A1 receptor the C6 alkyl substituent could extend

M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
2585
the coiling of residues Leu-58, Ala-59, Ile-60, Pro-61
and Phe-62. The N-alkyl pockets on the two receptors
therefore correspond to very similar areas but the coil-
ing in this region for the A2A is much more relaxed
accommodating up to 4 alkyl carbons.
prepared following known methods.12 The 2-bromo-N-
alkyl-propanamides were prepared from 2-bromo-
propanoyl bromide. The syntheses of the primary amides
5a, 6a, 7a and 8a have been previously reported.12
The ®nal region to be investigated was that around the
phenyl ring. For both the A1 and A2A receptors amino
acid His-278 was seen to interact with the phenyl group.
In both cases the histidine imidazole ring was almost
parallel to the aromatic ring with a `face-on' arrange-
ment, thereby maximizing p orbital interactions. This
His-278 residue has previously been implicated in ligand
ꢀ-(4-Mercapto-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)amides (5). The following general procedure was
used: 2-bromo-N-ethyl-propanamide (1.60 g, 8.84 mmol)
was added to a solution of 1-phenylpyrazolo[3,4-d]pyr-
imidine-4,6(5H,7H)-dithione (4) (2.8 g, 0.011 mol) in
pyridine (40 mL). The solution was stirred at room
temperature for 3 h. Treatment with ethyl acetate (30
mL) resulted in precipitation of a white solid. The product
was recrystallized from DMSO and water to a€ord a-(4-
mercapto-1-phenylpyrazolo[3,4-d] pyrimidin-6-ylthio)-N-
ethyl-propanamide (5b). Yield 2.53 g, 80%; mp dec 272±
19
binding from mutagenesis studies for both the A1 and
15
A2A receptors. For the A1 receptor the ligand phenyl
ring is further bounded by the hydrophobic residues
Val-58 and Leu-98. In the A2A model the opposite face
of the phenyl ring (to the histidine) is ¯anked by
hydrophobic residues Ile-274 and Val-275 of helix VII,
giving an indication of how the phenyl ring juts into the
coils of helix VII with the imidazole side chain of His-
278 emerging from the helix to meet it.
276 ꢀC. H NMR (200 MHz, DMSO-d6) d 0.94 (t, 3H,
1
J=7.2 Hz, CH2CH3), 1.58 (d, 3H, J=7.1 Hz, CH3),
3.03 (m, 2H, J=5.9 Hz, 7.1 Hz, CH2CH3), 4.46 (q, 1H,
0
J=7.0 Hz, SCH), 7.42 (t, 1H, J=7.4 Hz, C4 Harom),
0
0
7.59 (t, 2H, J=8.1 Hz, 7.4 Hz, C3 , C5 Harom), 8.06 (d,
0
0
2H, J=8.2 Hz, C2 , C6 Harom), 8.21 (br t, 1H, J=5.3 Hz,
NH), 8.36 (s, 1H, C3H), 14.10 (br s, 1H, N5H). 13C
NMR (62.8 MHz, DMSO-d6) d 14.1 (q, CH2CH3), 18.2
(q, CH3), 33.7 (t, CH2CH3), 44.7 (d, SCH), 116.4 (s,
As an overall view of the ligand binding site models, the
ligand in the A1 receptor sits in the pore formed by the
helices in much closer contact with helices II and III
than the others. The phenyl ring comes out almost at a
right angle to the bicyclic nucleus, which itself lies in
approximately the same plane as helices II and III. At
the A2A receptor the pyrazolo-pyrimidine core occupies
a much more central position within the pore than at
the A1. The phenyl ring comes down and outwards
towards helix VII while the C6 substituents are directed
into the coils of helix II. The C4 thiomethyl substituent
points directly from that central position into helix III.
0
C3a), 121.3 (d, C2 , C6 ), 127.1 (s, C4 ), 129.3 (d, C3 , C5 ),
0
0
0
0
0
138.0 (s, C1 ), 138.1 (d, C3), 146.7 (s, C7a), 160.1 (s, C6),
169.6 (s, CˆO), 180.5 (s, C4). IR (KBr-disc) nmax 3325,
NH; 3125, NH; 1650, CˆO; 1600 cm 1, CˆC. Anal.
calcd for (C16H17N5OS2): C, 50.8; H, 4.2; N, 21.3;
found: C, 50.7; H, 3.9; N, 21.4%.
ꢀ-(4-Mercapto-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-propyl-propanamide (5c). Yield 80%; mp dec
257±259.5 ꢀC. 1H NMR (200 MHz, DMSO-d6) d 0.75 (t,
3H, J=7.3 Hz, CH2CH2CH3), 1.33 (sextet, 2H, J=7.0
Hz, CH2CH2CH3), 1.57 (d, 3H, J=7.0 Hz, CH3), 2.94
(m, 2H, J=5.8 Hz, 7.0 Hz, CH2CH2CH3), 4.46 (q, 1H,
Experimental
0
J=7.0 Hz, SCH), 7.41 (t, 1H, J=7.3 Hz, C4 Harom),
Melting points were determined on a Gallenkamp melt-
1
ing point apparatus and are uncorrected. H and 13C
0
7.57 (t, 2H, J=7.7 Hz, C3 , C5 Harom), 8.05 (d, 2H,
0
0
0
NMR spectra were obtained on Bruker WM-250, Bruker
CXP 300 and Varian Gemini-200. The type of carbon
atom was assigned by using the DEPT pulse sequence
obtained on a Varian Gemini-200; q=methyl, t=methyl-
ene, d=methine, and s=quaternary. COSYs and
HMQCs were run on a Varian Unity 400 MHz instru-
ment. Unless otherwise stated, DMSO-d6 was used as a
solvent and the solvent peak was used as the internal
standard. IR spectra were recorded as KBr discs on
Jasco IR-810 or Perkin-Elmer FTIR 1700 spectro-
meters. Starting materials were obtained from Aldrich
Pty Ltd. Ethanol was dried by re¯ux and distillation
over magnesium turnings and a catalytic amount
of iodine and was stored over 3 A molecular sieves.
Pyridine was dried by re¯ux and distilled from potas-
sium hydroxide and stored over 4 A molecular sieves.
DMF was dried over BaO. Hexane was distilled prior
to use.
J=7.6 Hz, C2 , C6 Harom), 8.24 (br t, 1H, J=5.5 Hz,
NH), 8.35 (s, 1H, C3H), 14.08 (br s, 1H, N5H). 13C
NMR (62.8 MHz, DMSO-d6) d 11.0 (q, CH2CH2CH3),
18.3 (q, CH3), 21.9 (t, CH2CH2CH3), 39.2 (t, CH2CH2
0
CH3), 44.7 (d, SCH), 116.3 (s, C3a), 121.3 (d, C2 , C6 ),
0
0
0
0
0
127.1 (s, C4 ), 129.3 (d, C3 , C5 ), 138.0 (s, C1 ), 138.1 (d,
C3), 146.4 (s, C7a), 160.1 (s, C6), 169.8 (s, CˆO), 180.1
(s, C4). IR (KBr-disc) nmax 3325, NH; 1650, CˆO; 1600
cm 1, CˆC. Anal. calcd for (C17H19N5OS2): C, 54.7; H,
5.1; N, 18.8; found: C, 54.6; H, 5.2; N, 18.7.
ꢀ-(4-Mercapto-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-butyl-propanamide (5d). Yield 85%; mp dec 255±
261 ꢀC. H NMR (200 MHz, DMSO-d6) d 0.79 (t, 3H,
J=7.0 Hz, CH2CH2CH2CH3), 1.34 (m, 4H, J=7.0 Hz,
CH2CH2CH2CH3), 1.58 (d, 3H, J=7.2 Hz, CH3), 3.00
(m, 2H, J=6.9 Hz, 5.8 Hz, CH2CH2CH2CH3), 4.48 (q,
1
0
1H, J=7.0 Hz, SCH), 7.42 (t, 1H, J=7.3 Hz, C4
0
Harom), 7.57 (t, 2H, J=7.8 Hz, C3 , C5 Harom), 8.06 (d,
0
0
0
The starting compounds, (ethoxymethylene)malono-
nitrile, 1-phenyl-5-amino-4-cyanopyrazole, and 1-phenyl-
pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dithione (4) were
2H, J=7.7 Hz, C2 , C6 Harom), 8.22 (br t, 1H, J=5.5 Hz,
NH), 8.36 (s, 1H, C3H), 14.09 (br s, 1H, N5H). 13C
NMR (50.3 MHz, DMSO-d6) d 13.6 (q, CH2CH2

2586
M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
0
0
0
CH2CH3), 18.4 (q, CH3), 19.5 (q, CH2CH2CH2CH3),
31.0 (t, CH2CH2CH2CH3), 38.6 (t, CH2CH2CH2CH3),
C5 Harom), 8.15 (d, 2H, J=8.1 Hz, C2 , C6 Harom), 8.20
(br t, 1H, J=5.5 Hz, NH), 8.50 (s, 1H, C3H). 13C NMR
(62.8 MHz, DMSO-d6) d 11.1 (q, CH2CH2CH3), 11.5
(q, SCH3), 18.3 (q, CH3), 22.0 (t, CH2CH2CH3), 39.2 (t,
CH2CH2CH3), 44.3 (d, SCH), 110.5 (s, C3a), 120.9 (d,
0
0
44.8 (d, SCH), 116.4 (s, C3a), 121.3 (d, C2 , C6 ), 127.1 (s,
0
0
0
0
C4 ), 129.4 (d, C3 , C5 ), 138.0 (s, C1 ), 138.2 (d, C3), 146.4
(s, C7a), 160.1 (s, C6), 169.7 (s, CˆO), 180.0 (s, C4). IR
(KBr-disc) nmax 3225, NH; 3125, NH; 1650, CˆO; 1600
cm 1, C=C. Anal. calcd for (C18H21 N5OS2): C, 55.8; H,
5.5; N, 18.1; found: C, 55.9; H, 5.5; N, 17.8%.
0
0
0
0
0
C2 , C6 ), 126.8 (s, C4 ), 129.4 (d, C3 , C5 ), 133.8 (d, C3),
0
138.2 (s, C1 ), 151.0 (s, C7a), 165.8 (s, C4), 168.0 (s, C6),
170.5 (s, CˆO). IR (KBr-disc) nmax 3310, NH; 3100,
NH; 1660, CˆO; 1600 cm 1, C=C. Anal. calcd for
(C18H21N5OS2): C, 55.8; H, 5.5, N, 18.1; found: C, 55.7;
H, 5.2; N, 18.3%.
ꢁ-(4-Mercapto-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-cyclopentyl-propanamide (5e). Yield 84%; mp
dec 260±269 ꢀC. 1H NMR (200 MHz, DMSO-d6) d
1.14±1.75 (m, 8H, 4ÂCH2 of cyclopentyl ring), 1.58 (d,
ꢀ-(4-Methylthio-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-butyl-propanamide (6d). Yield 70%; mp 194±
196 ꢀC. H NMR (250.12 MHz, DMSO-d6) d 0.75 (t,
3H, J=7.2 Hz, CH2CH2CH2CH3), 1.22 (m, 4H, J=7.2
Hz, CH2CH2CH2CH3), 1.55 (d, 3H, J=7.0 Hz, CH3),
2.69 (s, 3H, SCH3), 3.00 (m, 2H, J=7.0 Hz, 6.2 Hz,
CH2CH2CH2CH3), 4.50 (q, 1H, J=7.0 Hz, SCH), 7.38
0
3H, J=7.0 Hz, CH3), 3.92 (m, 2H, J=6.8 Hz, C1 H),
1
4.48 (q, 1H, J=7.0 Hz, SCH), 7.42 (t, 1H, J=7.4 Hz,
00
00
C4 Harom), 7.60 (t, 2H, J=8.1 Hz, 7.7 Hz, C3
00
00
00
C5 Harom), 8.06 (d, 2H, J=7.6 Hz, C2 , C6 Harom), 8.35
(br d, 1H, J=7.0 Hz, NH), 8.35 (s, 1H, C3H), 14.06 (br
s, 1H, N5H). 13C NMR (75.5 MHz, DMSO-d6) d 18.3
0
0
0
0
0 0
(t, 1H, J=8.0 Hz, C4 Harom), 7.57 (t, 2H, J=7.8 Hz, C3 ,
(q, CH3), 23.4 (t, C3 , C4 ), 31.8, 31.9 (t, C2 , C5 ), 44.7 (d,
0
00
SCH), 50.7 (d, C1 H), 116.3 (s, C3a), 121.2 (d, C2 , C6 ),
00
0
0
0
C5 Harom), 8.13 (d, 3H, J=8.4 Hz, C2 , C6 Harom, NH),
8.49 (s, 1H, C3H). 13C NMR (50.3 MHz, DMSO-d6) d
11.7 (q, SCH3), 13.6 (q, CH2CH2CH2CH3), 18.4 (q,
CH3), 19.4 (t, CH2CH2CH2CH3), 31.0 (t, CH2CH2
CH2CH3), 38.5 (t, CH2CH2CH2CH3), 44.4 (d, SCH),
00
00
00
00
127.0 (s, C4 ), 129.3 (d, C3 , C5 ), 137.9 (s, C1 ), 138.1 (d,
C3), 146.3 (s, C7a), 160.1 (s, C6), 169.1 (s, CˆO), 179.9
(s, C4). IR (KBr-disc) nmax 3275, NH; 3100, NH; 1650,
CˆO; 1600 cm 1, C=C. Anal. calcd for (C19H21
N5OS2): C, 57.1; H, 5.3; N, 17.5; found: C, 57.4; H, 5.2;
N, 17.3%.
0
0
0
110.5 (s, C3a), 120.8 (d, C2 , C6 ), 126.7 (s, C4 ), 129.4 (d,
0
0
C3 , C5 ), 133.8 (d, C3), 138.2 (s, C1 ), 150.9 (s, C7a),
0
165.7 (s, C4), 167.9 (s, C6), 170.4 (s, CˆO). IR (KBr-
1
ꢀ-(4-Methylthio-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)amides (6). The following general procedure was
used: d-(4-mercapto-1-phenylpyrazolo[3,4-d]pyrimidin-
6-ylthio)N-ethyl-propanamide (2.00 g 5.57 mmol) was
added to a sodium hydroxide solution (1.5 M, 20 mL).
Iodomethane (0.7 mL, 1.6 g, 0.011 mol) was added, and
the reaction mixture stirred at RT for 30 min. A white
solid precipitated, was collected, washed with water and
recrystallized from DMSO and water to a€ord a-(4-
methylthio-1-phenylpyrazolo[3,4-d] pyrimidin-6-ylthio)-
N-ethyl-propanamide (6b). Yield 1.7 g, 82%; mp 211±
disc) nmax 3275, NH; 3050, NH; 1675, CˆO; 1650 cm
,
CˆO. Anal. calcd for (C19H23N5OS2): C, 56.8; H, 5.8;
N, 17.4; found: C, 56.8; H, 5.6; N, 17.3%.
ꢀ-(4-Methylthio-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-cyclopentyl-propanamide (6e). Yield 76%; mp
207±210 ꢀC. 1H NMR (250.12 MHz, DMSO-d6) d 1.22±
1.78 (m, 8H, CH2 of cyclopentyl ring), 1.56 (d, 3H,
J=6.9 Hz, CH3), 2.70 (s, 3H, SCH3), 3.93 (m, 1H, J=6.6
Hz, 6.3 Hz, NC10H), 4.52 (q, 1H, J=6.8 Hz, SCH), 7.38
00
(t, 1H, J=7.4 Hz, C4 Harom), 7.59 (t, 2H, J=7.9 Hz,
212.5 ꢀC. H NMR (250.1 MHz, DMSO-d6) d 0.93 (t,
1
00
C3 , C5 Harom), 8.09 (br d, 1H, 6.7 Hz, NH), 8.14 (d,
00
00
00
3H, J=7.2 Hz, CH2CH3), 1.54 (d, 3H, J=7.1 Hz, CH3),
2.68 (s, 3H, SCH3), 3.03 (m, 2H, J=5.6 Hz, 7.1 Hz,
CH2CH3), 4.48 (q, 1H, J=7.0 Hz, SCH), 7.37 (t, 1H,
3H, J=7.6 Hz, C2 , C6 Harom, NH), 8.49 (s, 1H, C3H).
13C NMR (75.5 MHz, DMSO-d6 at 60 ꢀC) d 11.3 (q,
0
SCH3), 18.0 (q, CH3), 23.1 (t, C3 H2, C4 H2), 31.7, 31.8
0
0
J=7.4 Hz, C4 Harom), 7.57 (t, 2H, J=8.3 Hz, 7.5 Hz,
0 0
(t, C2 H2, C5 H2 interchangeable), 43.9 (d, SCH), 50.4
0
0
0
0
0 00 00
(d, C1 H), 110.2 (s, C3a), 120.6 (d, C2 , C6 ), 126.4 (s,
C3 , C5 Harom), 8.12 (d, 2H, J=8.7 Hz, C2 , C6 Harom),
8.22 (br t, 1H, J=5.0 Hz, NH), 8.46 (s, 1H, C3H). 13C
NMR (62.8 MHz, DMSO-d6) d 11.5 (q, SCH3), 14.3 (q,
CH2CH3), 18.3 (q, CH3), 33.7 (t, CH2CH3), 44.2 (d,
00
00
00
00
C4 ), 129.0 (d, C3 , C5 ), 133.4 (d, C3), 137.9 (s, C1 ),
150.7 (s, C7a), 165.3 (s, C4), 167.7 (s, C6), 169.6 (s,
CˆO); IR (KBr-disc) nmax 3275, NH; 3050, NH; 1650,
CˆO; 1600 cm 1, C=C. Anal. calcd for (C20H23
N5OS2): C, 58.1; H, 5.6; N, 16.9; found: C, 58.1; H, 5.5;
N, 16.7%.
0
SCH), 110.5 (s, C3a), 120.9 (d, C2 , C6 ), 126.8 (s, C4 ),
0
0
0
0
0
129.4 (d, C3 , C5 ), 133.8 (d, C3), 138.2 (s, C1 ), 151.0 (s,
C7a), 165.8 (s, C4), 167.9 (s, C6), 170.3 (s, CˆO). IR
(KBr-disc) nmax 3320, NH; 3080, NH; 1655, CˆO; 1600,
cm 1 C=C. Anal. calcd for (C17H19N5OS2): C, 54.7; H,
5.1; N, 18.8; found: C, 54.3; H, 5.5; N, 18.5%.
ꢀ-(4-Methylthio-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N,N-diethyl-propanamide (6f). Yield 60%; mp 108±
109 ꢀC. H NMR (200 MHz, DMSO-d6) d 1.00 (t, 3H,
1
ꢀ-(4-Methylthio-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl-
thio)N-propyl-propanamide (6c). Yield 82%; mp 195±
J=7.1 Hz, CH2CH3), 1.11 (t, 3H, J=7.1 Hz, CH2CH3),
1.58 (d, 3H, J=6.8 Hz, CH3), 2.69 (s, 3H, SCH3), 3.31
(m, 4H, 2ÂCH2, J=7.1 Hz, 6.5 Hz, 2ÂCH2CH3), 5.06
(q, 1H, J=6.9 Hz, SCH), 7.39 (t, 1H, J=7.4 Hz,
196.3 ꢀC. H NMR (250.12 MHz, DMSO-d6) d 0.74 (t,
1
3H, J=7.4 Hz, CH2CH2CH3), 1.34 (sextet, 2H, J=7.2
Hz, CH2CH2CH3), 1.55 (d, 3H, J=7.0 Hz, CH3), 2.69
(s, 3H, SCH3), 2.98 (m, 2H, J=6.0 Hz, 7.3 Hz,
CH2CH2CH3), 4.50 (q, 1H, J=7.0 Hz, SCH), 7.38 (t,
0
C4 Harom), 7.58 (t, 2H, J=7.8 Hz, C3 , C5 Harom), 8.10
0
0
(d, 2H, J=7.6 Hz, C2 , C6 Harom), 8.48 (s, 1H, C3H). 13
C
0
0
NMR (50.3 MHz, DMSO-d6) d 11.6 (q, SCH3), 12.7 (q,
CH2CH3), 14.7 (q, CH2CH3), 19.4 (q, CH3), 40.4 (t,
0
0
1H, J=7.4 Hz, C4 Harom), 7.58 (t, 2H, J=7.9 Hz, C3 ,

M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
2587
0
2ÂCH2CH3), 42.0 (d, SCH), 110.5 (s, C3a), 120.9 (d, C2 ,
NH2), 8.01 (br t, 1H, J=5.5 Hz, NHamide), 8.16 (d, 2H,
J=8.2 Hz, C2 , C6 Harom), 8.27 (s, 1H, C3H). 13C NMR
(50.3 MHz, DMSO-d6) d 13.6 (q, CH2CH2CH2CH3),
18.2 (q, CH3), 19.4 (t, CH2CH2CH2CH3), 31.0 (t,
CH2CH2CH2CH3), 38.4 (t, CH2CH2CH2CH3), 43.1 (d,
0
C6 ), 126.8 (s, C4 ), 129.2 (d, C3 , C5 ), 133.7 (d, C3),
0
0
0
0
0
0
138.0 (s, C1 ), 150.9 (s, C7a), 165.9 (s, C4), 167.2 (s, C6),
169.5 (s, CˆO). IR (KBr-disc) nmax 1660, CˆO; 1600
cm 1, CˆC. Anal. calcd for (C19H23N5OS2): C, 56.8; H,
5.8; N, 17.4; found: C, 57.1; H, 5.8; N, 17.5%.
0
SCH), 99.4 (s, C3a), 120.4 (d, C2 , C6 ), 126.0 (s, C4 ),
0
0
0
0
0
129.2 (d, C3 , C5 ), 134.4 (d, C3), 138.9 (s, C1 ), 153.5 (s,
C7a), 157.4 (s, C4), 168.7 (s, C6), 170.9 (s, CˆO). IR
(KBr-disc) nmax 3525, NH; 3300, NH; 3050, NH; 1660
CˆO; 1595 cm 1, CˆC. Anal. calcd for (C18H22N6OS):
C, 58.4; H, 6.0; N, 22.7; found: C, 58.2; H, 6.3; N, 22.9%.
ꢀ-(4-Amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)
amides (7). The following general procedure was used:
a saturated solution of ammonia in ethanol (15 mL) was
added to a-(4-methylthio-1-phenylpyrazolo[3,4-d] pyr-
imidin-6-ylthio)-N-ethyl-propanamide (0.17 g, 0.44
mmol). The solution was heated at 100 ꢀC for 72 h in a
bomb. The solvent was removed to yield a white solid.
This was recrystallized from DMSO and water to a€ord
a-(4-amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)-
N-ethyl-propanamide (7b). Yield 0.12 g, 79%; mp dec
ꢀ-(4-Amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)-
N-cyclopentyl-propanamide (7e). Yield 80%; mp dec
252±257 ꢀC. H NMR (300.1 MHz, DMSO-d6) d 1.05±
1
1.70 (m, 8H, 4ÂCH2 of cyclopentyl group), 1.49 (d, 4H,
J=7.0 Hz, CH3), 1.48 (d, 3H, J=7.0 Hz, CH3), 3.92 (m,
261±264 ꢀC. H NMR (200 MHz, DMSO-d6) d 0.94 (t,
1H, J=7.1 Hz, 5.9 Hz, C1 H), 4.39 (q, 1H, J=7.0 Hz,
1
0
3H, J=7.2 Hz, CH2CH3), 1.53 (d, 3H, J=7.0 Hz, CH3),
3.05 (m, 2H, J=6.0 Hz, 7.0 Hz, CH2CH3), 4.41 (q, 1H,
0
J=7.1 Hz, SCH), 7.35 (t, 1H, J=7.4 Hz, C4 ,Harom),
SCH), 7.32 (t, 1H, J=7.4 Hz, C400Harom), 7.55 (t, 2H,
J=7.8 Hz, C300, C500Harom), 7.98 (br d, 2H, J=7.3 Hz,
NHamide, NH), 8.09 (br s, 1H, NH), 8.12 (d, 2H, J=8.1
Hz, C200, C600Harom), 8.27 (s, 1H, C3H). 13C NMR (75.5
0
7.56 (t, 2H, J=7.7 Hz, C3 , C5 Harom), 8.19 (d, 2H,
0
0
0
0
0
J=8.4 Hz, C2 , C6 Harom), 7.99 (br s, 2H, NH2), 8.11 (br
t, 1H, J=5.4 Hz, NH), 8.29 (s, 1H, C3H). 13C NMR
(62.8 MHz, DMSO-d6) d 14.2 (q, CH2CH3), 18.1 (q,
CH3), 33.6 (t, CH2CH3), 43.1 (d, SCH), 99.3 (s, C3a),
MHz, DMSO-d6) d 17.6 (q, CH3), 23.1 (t, C3 H2, C4 H2),
0
31.7, 31.8 (t, C2 H2, C5 H2 interchangeable), 42.5 (d,
00
SCH), 50.3 (d, C1 H), 99.1 (s, C3a), 120.3 (d, C2 , C6 ),
00
125.8 (s, C4 ), 129.0 (d, C3 , C5 ), 134.1 (d, C3), 138.6 (s,
0
0
00
00
00
0
0
0
0
0
120.4 (d, C2 , C6 ), 126.0 (s, C4 ), 129.2 (d, C3 , C5 ), 134.4
00
C1 ), 153.2 (s, C7a), 157.2 (s, C4), 168.6 (s, C6), 170.2 (s,
0
(d, C3), 138.9 (s, C1 ), 151.1 (s, C7a), 157.5 (s, C4), 168.8
CˆO). IR (KBr-disc) nmax 3325, NH; 3250, NH; 3175,
1
(s, C6), 170.8 (s, CˆO). IR (KBr-disc) nmax 3250, NH;
3190, NH; 1665, CˆO; 1600 cm 1, CˆC. Anal. calcd
for (C16H18N6OS): C, 56.1; H, 5.3; N, 24.5; found: C,
56.1; H, 5.4; N, 24.4%.
NH; 1660 CˆO; 1595 cm
CˆC. Anal. calcd for
(C19H22N6OS): C, 59.7; H, 5.8; N, 22.0; found: C, 59.6;
H, 5.9; N, 22.2%.
ꢀ-(4-Amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)-
N,N-diethyl-propanamide (7f). Yield 65%; mp dec 222±
ꢀ-(4-amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)-
N-propyl-propanamide (7c). Puri®ed by chromato-
graphy on silica gel (230±400 mesh, 60 A) using ethyl
acetate:hexane (1:1) then ethyl acetate (100%), Rf (ethyl
225 ꢀC. H NMR (200 MHz, DMSO-d6) d 0.98 (t, 3H,
1
J=7.0 Hz, CH2CH3), 1.11 (t, 3H, J=7.0 Hz, CH2CH3),
1.54 (d, 3H, J=6.6 Hz, CH3), 3.35 (m, 4H, J=7.2 Hz,
6.8 Hz, 2ÂCH2CH3), 5.02 (q, 1H, J=6.9 Hz, SCH),
acetate, 100%)=0.75. Yield 79%; mp 248±249 ꢀC. H
1
0
NMR (200 MHz, DMSO-d6) d 0.72 (t, 3H, J=7.3 Hz,
CH2CH2CH3), 1.29 (sextet, 2H, J=7.1 Hz, CH2CH2
CH3), 1.50 (d, 3H, J=6.9 Hz, CH3), 2.96 (br q, 2H,
J=6.0 Hz, 6.9 Hz, CH2CH2CH3), 4.43 (q, 1H, J=7.0
7.32 (t, 1H, J=7.4 Hz, C4 Harom), 7.57 (t, 2H, J=7.3
0
0
Hz, C3 , C5 Harom), 7.99 (br s, 1H, NH), 8.16 (d, 3H,
J=8.5 Hz, C2 , C6 Harom, NH), 8.29 (s, 1H, C3H). 13C
NMR (50.3 MHz, DMSO-d6) d 12.8 (q, CH2CH3), 14.8
(q, CH2CH3), 19.8 (q, CH3), 39.2 (t, CH2CH3), 40.0 (t,
0
0
0
Hz, SCH), 7.32 (t, 1H, J=7.3 Hz, C4 Harom), 7.54 (t,
0
0
0
CH2CH3), 42.0 (d, SCH), 99.5 (s, C3a), 120.5 (d, C2 ,
2H, J=7.9 Hz, C3 , C5 Harom), 7.94 (br s, 2H, NH2),
8.07 (br t, 1H, J=5.5 Hz, NHamide), 8.16 (d, 2H, J=7.4
0
C6 ), 126.2 (s, C4 ), 129.2 (d, C3 , C5 ), 134.4 (d, C3),
0
0
0
Hz, C2 , C6 Harom), 8.26 (s, 1H, H3). 13C NMR (62.8
MHz, DMSO-d6) d 11.0 (q, CH2CH2CH3), 18.0 (q,
CH3), 39.8 (t, CH2CH2CH3), 43.0 (d, SCH), 99.3 (s,
0
C3a), 120.5 (d, C2 , C6 ), 126.0 (s, C4 ), 129.2 (d, C3 , C5 ),
0
134.4 (d, C3), 138.9 (s, C1 ), 153.6 (s, C7a), 157.5 (s, C4),
138.9 (s, C1 ), 153.5 (s, C7a), 157.6 (s, C4), 168.0 (s, C6),
0
0
0
170.1 (s, CˆO). IR (KBr-disc) nmax 3325, NH; 3150,
NH; 1660, CˆO; 1635, CˆO; 1600 cm 1, CˆC. Anal.
calcd for (C18H22N6OS): C, 58.4; H, 6.0; N, 22.7; found:
C, 58.2; H, 6.0; N, 23.1%.
0
0
0
0
168.8 (s, C6), 171.0 (s, CˆO). IR (KBr-disc) nmax 3525,
NH; 3325, NH; 3150, NH; 3100, NH; 1665, CˆO; 1600
cm 1, CˆC. Anal. calcd for (C17H20N6OS): C, 57.3; H,
5.7; N, 23.6; found: C, 57.2; H, 5.6; N, 23.4%.
ꢀ-{6-(10 -N-Ethylcarbamoylethylthio)-1-phenylpyrazolo
[3,4-d]pyrimidin-4-ylthio}-N-ethyl-propanamide (8b). 2-
Bromo-N-ethyl-propanamide (1.19 g, 0.007 mol) was
added to a solution of 1 - phenylpyrazolo[3,4 - d]pyr-
imidine - 4,6(5H,7H)-dithione (0.67 g, 0.0026 mol) in
sodium hydroxide solution (1 M, 6 mL). The mixture was
stirred at room temperature for 90 min. A white solid
precipitated and was recrystallized from DMSO and
water to yield a-{6-(10 -N-ethylcarbamoylethylthio)-1-
phenylpyrazolo [3,4-d]pyrimidin-4-ylthio}-N-ethyl-propa-
namide (8b). Yield 1.0 g, 85%; mp 191±192 ꢀC. Diaster-
eomersÐall peaks are duplicated: 1H NMR (250.1
ꢀ-(4-Amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-ylthio)-
N-butyl-propanamide (7d). Yield 75%; mp 213±216 ꢀC.
1H NMR (200 MHz, DMSO-d6) d 0.72 (t, 3H, J=7.3
Hz, CH2CH2CH2CH3), 1.21 (m, 4H, J=7.2 Hz, CH2
CH2CH2CH3), 1.48 (d, 3H, J=7.0 Hz, CH3), 2.98 (br q,
2H, J=6.5 Hz, 6.0 Hz, CH2CH2CH2CH3), 4.42 (q, 1H,
0
J=7.0 Hz, SCH), 7.31 (t, 1H, J=7.4 Hz, C4 Harom),
0
0
7.53 (t, 2H, J=7.7 Hz, C3 , C5 Harom), 7.93 (br s, 2H,

2588
M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
MHz, DMSO-d6)
d
0.95 (2Ât, 6H, J=7.2 Hz,
4ÂCH3), 19.1, 19.2 (t, 4ÂCH2CH2CH2CH3), 30.8 (t,
4ÂCH2CH2CH2CH3), 38.3, 38.4 (t, 4ÂCH2CH2CH2
CH3), 42.6 (d, 2ÂSCH), 43.9, 44.0 (d, 2ÂSCH), 109.9,
2ÂCH2CH3), 1.05 (2Ât, 6H, J=7.2 Hz, 2ÂCH2CH3),
1.58 (2Âd, 6H, J=7.0 Hz, 2ÂCH3), 3.08 (m, 4H, J=5.5
Hz, 7.1 Hz, 2ÂCH2CH3), 4.50 (2Âq, 1H, J=7.0 Hz,
C6SCH), 4.78 (2Âq, 1H, J=7.0 Hz, C4SCH), 7.38±8.18
(m, 5H, CHarom), 8.24 (2Âbr t, 1H, J=5.0 Hz, NH), 8.37
(2Âbr t, 1H, J=5.0 Hz, NH), 8.52 (s, 1H, C3H). 13C
NMR (62.8 MHz, DMSO-d6) d 14.2 (q, 4ÂCH2CH3),
18.1 (q, 2ÂCH3), 18.9 (q, 2ÂCH3), 33.6 (t, 2ÂCH2CH3),
33.7 (t, 2ÂCH2CH3), 42.7 (d, 2ÂSCH), 44.0, 44.1 (d,
0
0
0
110.0 (s, 2ÂC3a), 120.7 (d, C2 , C6 ), 126.5 (s, C4 ), 129.1
0
0
0
(d, C3 , C5 ), 133.5 (d, C3), 137.9 (s, C1 ), 150.8 (s, C7a),
164.0, 164.1 (s, 2ÂC4), 167.7, 167.8 (s, 2ÂC6), 169.8,
170.3 (s, 4ÂCˆO). IR (KBr-disc) nmax 3285, NH; 3100,
NH; 1650, CˆO; 1600 cm 1, CˆC. Anal. calcd for (C25
H34N6O2S2): C, 58.3; H, 6.7; N, 16.3; found: C, 58.7; H,
6.9; N, 16.2%.
0
0
0
2ÂSCH), 110.1 (s, C3a), 120.9 (d, C2 , C6 ), 126.8 (s, C4 ),
129.4 (d, C3 , C5 ), 133.8 (d, C3), 138.2 (s, C1 ), 151.0 (s,
C7a), 165.4 (s, C4), 166.0 (s, C6), 169.9, (s, 2ÂCˆO),
170.4 (s, 2ÂCˆO). IR (KBr-disc) nmax 3250, NH; 3080,
NH; 1675, CˆO; 1665, CˆO; 1655, CˆO; 1645, CˆO;
1600 cm 1, CˆC. Anal. calcd for (C21H26 N6O2S2): C,
55.0; H, 5.7; N, 18.3; found: C, 55.3; H, 5.8; N, 18.7%.
ꢀ-{6-(10-N-Cyclopentylcarbamoylethylthio)-1-phenylpyra-
zolo[3,4-d]pyrimidin-4-ylthio}-N-cyclopentyl-propanamide
(8e). Yield 72%; mp 186±188 ꢀC, 196±199 ꢀC. Diaster-
eoisomersÐall peaks are duplicated: 1H NMR (200
MHz, DMSO-d6) d 1.15±1.82 (m, 16H, 8ÂCH2 of
cyclopentyl groups), 1.57 (2Âd, 6H, J=6.9 Hz,
0
0
0
0
2ÂCH3), 3.97 (m, 2H, J=6.6 Hz, 2ÂC1 H), 4.50 (2Âq,
ꢀ-{6-(10 -N-Alkylcarbamoylethylthio)-1-phenylpyrazolo
[3,4-d]pyrimidin-4-ylthio}amides (8c±e). The following
general procedure was used for the remaining com-
pounds: 2-bromo-N-propyl-propanamide (3.04 g, 0.016
mol) was added to a solution of 1-phenylpyrazolo [3,4-d]
pyrimidine-4,6(5H,7H)-dithione (2.20 g, 0.0085 mol) in
sodium hydroxide (1.5 M, 20 mL) with 2-methylpropan-
2-ol (20 mL). The mixture was stirred at room tempera-
ture for 5 h. A white solid precipitated and was recrys-
tallized from DMSO and water to yield a-{6-(10 -N-
propylcarbamoylethylthio) - 1 - phenylpyrazolo [3,4 - d]
pyrimidin-4-ylthio}-N-propyl-propanamide (8c). Yield
2.6 g, 70%; mp 164.5±169 ꢀC. DiastereoisomersÐall
peaks are duplicated: 1H NMR (250.1 MHz, DMSO-d6)
d 0.73 (t, 6H, J=7.4 Hz, 2ÂCH2CH2CH3), 0.82 (2Ât,
6H, J=7.4 Hz, 2ÂCH2CH2CH3), 1.38 (m, 4H, J=7.3
Hz, 2ÂCH2CH2CH3), 1.58 (2Âd, 6H, J=6.9 Hz,
2ÂCH3), 3.00 (m, 4H, J=5.5 Hz, 6.9 Hz, 2ÂCH2CH3),
4.49 (2Âq, 1H, J=7.0 Hz, C6SCH), 4.79 (2Âq, 1H, J=6.9
Hz, C4SCH), 7.34±8.14 (m, 5H, CHarom), 8.21 (2Âbr t,
1H, NH), 8.35 (2Âbr t, 1H, J=5.1 Hz, NH), 8.48 (s, 1H,
H3). 13C NMR (62.8 MHz, DMSO-d6) d 10.6 10.7 (q,
2ÂCH2CH2CH3), 17.8, 18.0, 18.6, 18.7 (q, 4ÂCH3), 21.8
(t, 4ÂCH2CH2CH3), 40.5, 40.6 (t, 4ÂCH2CH2CH3), 42.7
(d, 2ÂSCH), 44.0, 44.1 (d, 2ÂSCH), 111.0 (s, C3a), 121.8
1H, J=6.9 Hz, C6SCH), 4.75 (2Âq, 1H, J=7.0 Hz,
C4SCH), 7.35±8.17 (m, 6H, J=7.0 Hz, 5CHarom, 1NH),
8.31 (2Âbr t, 1H, J=6.9 Hz, NH), 8.50 (s, 1H, H3). 13C
NMR (50.3 MHz, DMSO-d6) d 18.1, 18.4 (q, 2ÂCH3),
18.9, 19.1 (q, 2ÂCH3), 23.3, 23.4 (t, 8ÂCH2), 42.8, 42.9
(d, 2ÂSCH), 44.0, 44.2, (d, 2ÂSCH), 42.6 (d, 2ÂSCH),
0
00
50.6 (d, 2ÂC1 H), 100.0, 110.1 (s, 2ÂC3a), 120.8 (d, C2 ,
00
C6 ), 126.7 (s, C4 ), 129.4 (d, C3 , C5 ), 133.8 (d, C3),
00
00
00
00
138.0 (s, C1 ), 150.8 (s, C7a), 164.2, 164.3 (s, 2ÂC4),
168.0, 168.1 (s, 2ÂC6), 169.4, 169.9 (s, 4ÂCˆO). IR
(KBr-disc) nmax 3300, NH; 3100, NH; 1650, CˆO; 1600
cm 1, CˆC. Anal. calcd for (C27H34N6O2S2): C, 60.2;
H, 6.4; N, 15.6; found: C, 60.4; H, 6.5; N, 15.4%.
Preparation of membranes from rat cerebral cortex and
rat cerebellum
This followed a simpli®ed version of the Gray and
Whittaker method.12,20 Whole brains from Wistar rats
(25 rats, 300±350 g) were immersed in 0.32 M ice-cold
sucrose. The striata were explanted and placed in 10 mL
ice-cold bu€er (50 mM Tris±HCl (Sigma), 10 mM
MgCl2, pH 7.4), to be used for the preparation of A2
adenosine receptors. The cerebellum and remainder of
the cerebral cortex were placed in 15 mL of ice-cold
bu€er (50 mM Tris±HCl, 1 mM MgCl2, pH 7.4). The
tissue was homogenized (Brinkman polytron) and the
volume adjusted to 48.0 g. Homogenates were cen-
trifuged (1000 g for 10 min at 4 ꢀC) and the supernatant
(S1) recentrifuged (35,000 g for 15 min at 4 ꢀC). The
supernatant (S2) was discarded and every eight pellets
(P2) pooled and resuspended in ice-cold distilled water
(30 mL) and the volume was adjusted to 48.00 g. The
homogenates were recentrifuged (35,000 g for 15 min at
4 ꢀC). The pellets (P3) were pooled and resuspended in
ice-cold bu€er (60 mL of 50 mM Tris±HCl, 1 mM
MgCl2, pH 7.4). When stored at 30 ꢀC, binding to the
synaptosomal membranes remained unchanged for 1
month. The protein concentration was estimated
using Pierce BCA protein assay reagent using a modi®ed
Lowry protein assay.21 Tissue samples were solubilized
using 10 mL of 10% SDS and were made up to 100 mL.
The standards contained 0 to 120 mg of protein. Five
dilutions of tissue from 4 to 75% were sampled. The
absorbance was read using a Titertek Multiscan MC
0
0
0
0
0
(d, C2 , C6 ), 127.8 (s, C4 ), 130.4 (d, C3 , C5 ), 134.9 (d,
0
C3), 139.3 (s, C1 ), 152.3 (s, C7a), 165.8 (s, 2ÂC4), 169.5,
169.6 (s, 2ÂC6), 171.7, 172.2 (s, 4ÂCˆO). IR (KBr-disc)
1
nmax 3300, NH; 3125, NH; 1650, CˆO; 1600 cm
,
CˆC. Anal. calcd for (C23 H30N6O2S2): C, 56.8; H, 6.2;
N, 17.3; found: C, 57.2; H, 6.3; N, 17.2%.
ꢀ-{6-(10 -N-Butylcarbamoylethylthio)-1-phenylpyrazolo
[3,4-d]pyrimidin-4-ylthio}-N-butyl-propanamide (8d). Yield
72%; mp 148±149 ꢀC, 165±167 ꢀC. DiastereoisomersÐ
all peaks are duplicated: 1H NMR (250.1 MHz, DMSO-
d6) d 0.76 (t, 6H, J=7.3 Hz, 2ÂCH2CH2CH2CH3), 0.84
(2Ât, 6H, J=7.3 Hz, 2ÂCH2CH2CH2CH3), 1.29 (m,
8H, J=7.2 Hz, 2ÂCH2CH2CH2CH3), 1.57 (2Âd, 6H,
J=6.9 Hz, 2ÂCH3), 3.03 (m, 4H, J=7.4 Hz, 6.2 Hz,
2ÂCH2CH3), 4.50 (2Âq, 1H, J=7.0 Hz, C6SCH), 4.80
(2Âq, 1H, J=7.0 Hz, C4SCH), 7.36±8.16 (m, 6H,
5CHarom, NH), 8.28 (2Âbr t, 1H, J=5.5 Hz, NH), 8.50
(s, 1H, H3). 13C NMR (75.5 MHz, DMSO-d6) d 13.2
13.3 (q, 2ÂCH2CH2CH2CH3), 18.0, 18.1, 18.7, 18.8 (q,

M. Chebib et al. / Bioorg. Med. Chem. 8 (2000) 2581±2590
2589
Microtiter Plate Reader at 540 nm absorbance. The
average concentration of A1 tissue was 2.63 mg/mL.
37 ꢀC; ADA, EC 3.5.4.4) to remove endogenous adeno-
sine. Between 0.3 and 0.5 mg of protein per replicate
was incubated with 5 nM [3H]-CGS21680 (48.6 Ci/
mmol, 1 mCi/mL; New England Nuclear) for 95 min at
room temperature. Radioligand binding assays were
performed over 10 concentrations in duplicates. Each
compound was tested in two di€erent experiments.
Total volume of the A2A assay was 0.5 mL. The com-
pounds proved insoluble in bu€er and hence the stock
concentrations were made up in DMSO. The ®nal con-
centration of DMSO in the assay was 1 percent. The
assay was monitored against a control of total binding
and non-speci®c binding each containing 1 percent
DMSO. Non-speci®c binding was determined using cold
2-CADO (0.1 mM ®nal concentration). Reactions were
terminated by the addition of 3 mL of ice-cold incuba-
tion bu€er. This was ®ltered over presoaked (6 h)
Whatman GF/B Glass ®ber ®lters and washed with two
more 3 mL ice-cold bu€er washes. The ®lters were
transferred to vials, thoroughly shaken with 3 mL of
BCS scintillant ¯uid. Samples were equilibrated in the
dark for a minimum of 6 h before counting for 1 min in
a Packard Tricarb 2000CA series liquid scintillation
Analyzer at 40% eciency.
Preparation of membranes from rat striata
The bu€er was drained from the striata and weighed. The
striata were placed in 20 mL ice-cold bu€er (50 mM Tris±
HCl (Sigma), 10 mM MgCl2, pH 7.4) and homogenized
using the polytron, placed into centrifuge tubes and the
volume adjusted to 48.00 g, and centrifuged (35 000 g
for 15 min at 4 ꢀC). The supernatant was discarded and
the pellet resuspended in ice-cold bu€er (10 mL of 50
mM Tris±HCl, 10 mM MgCl2, pH 7.4). The homo-
genate was recentrifuged (35 000 g for 15 min at 4 ꢀC).
The supernatant was discarded and the pellets pooled
and resuspended in ice-cold bu€er (1 g of striata/10 mL
of 50 mM Tris±HCl, 10 mM MgCl2, pH 7.4). This was
stored at 30 ꢀC and remained unchanged for 1 month.
The protein concentration was estimated using Pierce
BCA protein assay reagent. The average concentration
of A2A tissue was 3.78 mg/mL.
[3H]-(R)-N6-(Phenylisopropyl)adenosine binding assay22
Aliquots of A1 tissue were thawed and preincubated
with adenosine deaminase (1 mg/mL for 20 min at
37 ꢀC; ADA, EC 3.5.4.4; Sigma) to remove endogenous
adenosine. Between 0.3 and 0.5 mg of protein per repli-
cate was incubated with 2 nM [3H]-(R)-N6-(phenyliso-
propyl) adenosine (60 Ci/mmol, 1 mCi/mL; New
England Nuclear) for 1 h at 37 ꢀC in an incubation
volume of 0.5 mL. Radioligand binding assays were
performed over 10 concentrations in duplicates. Each
compound was tested in two di€erent experiments.
Total volume of the A1 assay was 0.5 mL. The com-
pounds proved insoluble in bu€er and hence the stock
concentrations were made up in DMSO. The ®nal con-
centration of DMSO in the assay was 1%. The assay
was monitored against a control of total binding and
non-speci®c binding each containing 1% DMSO. Non-
speci®c binding was determined using cold CPA (0.1
mM ®nal concentration). Reactions were terminated by
the addition of 3 mL of ice-cold incubation bu€er. This
was ®ltered over Whatman GF/B Glass ®ber ®lters and
washed with two more 3-mL ice-cold bu€er washes. The
®lters were transferred to vials, thoroughly shaken with
3 mL of BCS scintillant ¯uid. Samples were equilibrated
in the dark for a minimum of 6 h before counting for 1
min in a Packard Tricarb 2000CA series liquid scintil-
lation Analyzer at 40% eciency.
The results from concentration±inhibition studies were
analyzed with non-linear-least-squares curve-®tting pro-
gram.23
A2AKi values were calculated using the Cheng
Pruso€ equation24 using the average Kd value of [3H]-
CGS21680 as 14.9 nM and a ®nal ligand concentration
of 5 nM. A2AKi values are geometric means from two
determinations, run in duplicateÆstandard error.
Acknowledgements
We thank the National Health and Medical Research
Council for support of this research.
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The results from concentration±inhibition studies were
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program.23 A1Ki values were calculated using the
Cheng±Pruso€ equation24 using the average Kd value of
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