High-pressure synthesis of enantiomerically pure C-6 substituted pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1016/S0960-894X(00)00620-X

The synthesis of enantiomerically pure C-6 substituted pyrazolo[3,4-d]pyrimidines has been performed by aromatic nucleophilic substitution of 4-amino-6-chloro-1-phenylpyrazolo[3,4-d]pyrimidine under conditions of high pressure at ambient temperature. Conventional synthetic conditions (reflux at atmospheric pressure) were unsuccessful. The S enantiomer 11 displayed higher affinity and selectivity for the adenosine A₁ receptor than the R enantiomer 12.

Full text of DOI:10.1016/S0960-894X(00)00620-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 191±193
High-Pressure Synthesis of Enantiomerically Pure
C-6 Substituted Pyrazolo[3,4-d]pyrimidines
Sally-Ann Poulsen,a David J. Youngb and Ronald J. Quinna,*
aAstraZeneca R&D Grith University, Brisbane, 4111, Australia
bFaculty of Science, Grith University, Brisbane, 4111, Australia
Received 18 September 2000; accepted 1 November 2000
AbstractÐThe synthesis of enantiomerically pure C-6 substituted pyrazolo[3,4-d]pyrimidines has been performed by aromatic
nucleophilic substitution of 4-amino-6-chloro-1-phenylpyrazolo[3,4-d]pyrimidine under conditions of high pressure at ambient
temperature. Conventional synthetic conditions (re¯ux at atmospheric pressure) were unsuccessful. The S enantiomer 11 displayed
higher anity and selectivity for the adenosine A1 receptor than the R enantiomer 12. # 2001 Elsevier Science Ltd. All rights
reserved.
Aromatic nucleophilic substitution at the C-2 position
of the nucleoside analogue 2-chloroadenosine to give
N-alkylated-2-aminoadenosines proceeds under rela-
tively harsh conditions.1 4 The general method is reac-
tion of 2-chloroadenosine with an excess of amine at
temperatures in excess of 100 ꢀC for extended periods.
Yields are variable and generally disappointing, how-
ever, the desirable biological properties of these com-
pounds as adenosine A2A receptor agonists has
accounted for acceptance of these poor yields. The
adenosine A2A selective agonist CGS21680 (1) is a
modi®ed 2-substituted adenosine.5
Substitution at the corresponding position in phenyl-
pyrazolo[3,4-d]pyrimidines resulted in a-[4-(methyl-
amino)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thiol]hex-
anamide (2) which has an A1 Ki of 0.745 nM and is
selective over the adenosine A2A receptor.6 Phenyl-
pyrazolo[3,4-d]pyrimidines having potency and selectivity
for the adenosine A1 receptor have been synthesised
following the general method as shown in Scheme 1.
This synthetic scheme introduces the C-6 substituent via
alkylation of sulfur in good yield. However, attempts in
our laboratory to prepare C-6 substituted pyrazolo[3,4-
d]pyrimidines with an enantiomerically pure centre a to
the C-6 sulfur failed due to rapid racemisation, pre-
sumably due to carbanion stabilisation by both sulfur
and the carbonyl. The corresponding compounds with a
nitrogen at C-6 would be expected to be more stable.
4-Amino-6-chloro-1-phenylpyrazolo[3,4-d]pyrimidine 5
was synthesised via a two-step synthesis (Scheme 2). The
one-pot reaction for formation of 4 from 4-amino-5-
cyano-1-phenylpyrazole with benzoyl isocyanate
proceeds through a urea intermediate, debenzoylation of
this intermediate with base, followed by cyclisation.7 5-
Amino-4-cyano-1-phenylpyrazole 3 (0.500 g, 2.7 mmol)
was sealed under an atmosphere of argon. A solution of
benzoyl isocyanate (0.600 g, 4.1 mmol) in 10 mL of dry
DMF was injected into the reaction vessel and the
reaction stirred for 12 h at 60 ꢀC. Ammonium hydroxide
(28%, 20 mL) was added to the reaction mixture, which
was then stirred for a further 12 h at 60 ꢀC. The solvent
was removed under reduced pressure, the crude product
was recrystallised from Me2SO and water, a€ording pure
48 in 73% yield. 4 (0.500 g, 2.20 mmol), phosphorous
*Corresponding author. Tel.: +61-7-3875-6000; fax: +61-7-3875-
6001; e-mail: r.quinn@az.gu.edu.au
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00620-X

192
S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 191±193
Scheme 1. General synthesis of 1-phenyl-4,6-disubstituted-pyrazolo[3,4-d]pyrimidines: (i) BrCHRCONH2, py; (ii) CH3I, NaOH (aq); (iii) NH3 (g),
EtOH, 110 ꢀC, 72 h.
Scheme 2. Synthetic route to 4-amino-6-chloro-1-phenylpyrazolo[3,4-d]pyrimidine (5): (i) PhCONCO, DMF, 60 ꢀC, 12 h; (ii) NH4OH, 60 ꢀC, 24 h,
73% overall; (iii) POCl3, PCl5, re¯ux, 40 h, 62%.
Scheme 3. Conventional and high-pressure synthetic methodology for target compounds 10±12: (i) Conventional: DMF, DIPEA, re¯ux, 4 days;
(ii) high pressure: 15 kbar, 40 ꢀC, DMF, DIPEA, 7 days.
oxychloride (10 mL, 0.11 mol) and phosphorous pen-
tachloride (2 g, 9.60 mmol) were re¯uxed for 40 h under
an atmosphere of argon. Excess phosphorous oxychlor-
ide was removed by distillation under reduced pressure,
leaving behind a yellow residue that solidi®ed on cool-
ing. Ice water (20 mL) was added to the solid residue
with vigorous stirring. The aqueous solution was then
extracted with diethyl ether (3 Â 15 mL). The ether
fractions were combined and dried over anhydrous
MgSO4, ®ltered, and the solvent removed, leaving a
yellow solid. The crude product was recrystallised from
Me2SO and water giving 5.9
nucleophiles such as primary amines.5 Compound 5 was
reacted with 3 equiv of the appropriate 2-aminopropan-
amides, 6 (racemic), 7 (S enantiomer), or 8 (R enantio-
mer), in DMF with an equivalent of base under a
pressure of 15 kbar at 40 ꢀC for 7 days (Scheme 3).12
The reaction mixtures were puri®ed by chromatography
to give the desired products 9 (racemic), 10 (S enantiomer)
and 11 (R enantiomer) each in 68% yield. Unreacted
starting material was the only other material recovered.
Table 1. Receptor binding results for rat A1 and A2A receptorsa
Compd
A1 receptor
Ki (nM)
A2A receptor
Ki (nM)
A2A Ki/A1 Ki
Application of the conventional strategy to the nucleo-
philic aromatic substitution of the pyrimidine ring of
4-amino-6-chloro-1-phenylpyrazolo[3,4-d]pyrimidine (6)
with 2-aminopropanamide (7) proved unsuccessful
(Scheme 3). None of the substituted product was detec-
ted, even after 4 days of reaction. Synthesis at high
pressure can now be carried out safely, and the method
can be used for increasing the rates of many types of
reactions, o€ering an alternative to synthesis at high
temperatures.10,11 In particular, synthesis at high pres-
sure has been reported to increase the rate of reaction of
nucleophilic aromatic substitutions, with neutral
9
10
11
Racemic
S
84.0Æ3.4
49.1Æ3.2
486Æ32
930Æ135
648Æ98
11
13
4
R
2120Æ339
aA1 receptor binding data utilising competitive displacement of speci®c
[3H]-N6-PIA binding from A1 receptors in whole rat brain membranes.
Data are the average of at least two independent experiments per-
formed in duplicate and expressed as KiÆSEM. Kd of [3H]-N6-PIA was
1 nM. A2A receptor binding data utilizing competitive displacement of
speci®c [3H]CGS21680 binding from A2A receptors in rat striatal
membranes. Data are the average of at least two independent experi-
ments performed in duplicate and expressed as KiÆSEM. Kd of
[3H]CGS21680 was 14.9 nM.

S.-A. Poulsen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 191±193
193
These results demonstrate the utility of high pressure as
an alternative to the use of elevated temperatures.
Reaction under high pressure has made available C-6
amino substituted pyrazolo[3,4-d]pyrimidines and may
o€er a more facile route to 2-substituted purines than
current methods. The results presented in Table 1
clearly highlight the stereochemical importance of the
C-6 substituent for anity to adenosine receptors. The
S enantoimer 11 had 10-fold higher anity for the A1
receptor and 3.3-fold higher anity for the A2A receptor
compared to the R enantiomer 12. The S enantiomer
was also more A1 selective (13-fold) than the less potent
R enantiomer (4-fold A1 selectivity).
1H, NH), 8.17 (s, 1H, H-3), 8.65 (br s, 1H, NH), 10.88 (br s,
1H, NH); 13C NMR (50 MHz, Me2SO-d6) d 92.6 (C-3a), 120.4
(C-20, C-60), 125.5 (C-40), 128.9 (C-30, C-50), 135.6 (C3), 139.1
(C-10), 153.6 (C-4), 156.2 (C-7a), 157.6 (C-6); IR (KBr disc)
3330 (1ꢀ amine NH); 3113 (CHaromatic); 1670 cm (CˆO).
1
9. 5 (Yield 62%); 1H NMR (200 MHz, Me2SO-d6) d 7.39±8.07
(m, 5H, H-40, H-30, H-50, H-20, H-60), 8.35 (br s, 1H, NH), 8.38
(s, 1H, H-3), 8.49 (br s, 1H, NH); 13C NMR (50 MHz, Me2SO-
d6) d 100.4 (C-3a), 121.0 (C-20, C-60), 126.7 (C-40), 129.3 (C-30,
C-50), 134.4 (C3), 138.4 (C-10), 153.9 (C-7a), 158.1 (C-4 or C-6),
158.8 (C-4 or C-6); IR (KBr disc) 3441, 3298 (1ꢀ amine NH);
1
3114 cm
(M+1[H]); ESMS (NI) 244.16. Calcd for (M±1[H]).
(CHaromatic); ESMS (PI) 246.17. Calcd for
10. Matsumoto, K.; Sera, A.; Uchida, T. Synthesis 1984, 1.
11. Isaacs, N. S.; George, A. V. Chem. Britain 1987, 23, 47.
12. Experimental. 5 (0.050 g, 0.21 mmol) and each of 6±8
(0.060 g, 0.68 mmol) were dissolved in dry DMF (1.6 mL,
0.57 mmol). N,N-Diisopropylethylamine (0.1 mL) was added
and the reaction mixtures transferred to 2 mL polyethylene
bulbs. The bulbs were sealed with a brass clamp (excluding
most of the air) and placed inside a Te¯on reaction vessel,
which was then ®lled with a mixture of castor oil/methanol
(85%:15%). The reaction vessel was placed in a PSIKA Pres-
sure Systems Limited Piston Cylinder High Pressure Reactor
and the reaction carried out at 15 kbar, 40 ꢀC for 7 days.
Puri®cation by chromatography (95% ethyl acetate/5%
methanol) gave yields of 68%. In addition to standard
characterisation, circular dichroism spectral data was recorded
Acknowledgements
The award of an Australian Postgraduate Award to
S.-A. P. is acknowledged. We acknowledge the support
of this work by the National Health and Medical
Research.
References and Notes
1. Marumoto, R.; Yoshioka, Y.; Miyashita, O.; Shima, S.;
Imai, K.-I.; Kawazoe, K.; Honjo, M. Chem. Pharm. Bull.
1975, 23, 759.
2. Trivedi, B. K. Nucleosides Nucleotides 1988, 7, 393.
3. Trivedi, B. K.; Bruns, R. F. J. Med. Chem. 1989, 32, 1667.
4. Francis, J. E.; Webb, R. L.; Ghai, G. R.; Hutchison, A. J.;
Moskal, M. A.; de Jesus, R.; Yokoyama, R.; Rovinski, S. L.;
Contardo, N.; Dotson, R.; Barclay, B.; Stone, G. A.; Jarvis,
M. F. J. Med. Chem. 1991, 34, 2570.
5. Jarvis, M. F.; Schulz, R.; Hutchison, A. J.; Do, U. H.; Sills,
M. A.; Williams, M. J. Pharmacol. Exp. Ther. 1989, 251, 88.
6. Poulsen, S.-A.; Quinn, R. J. J. Med. Chem. 1996, 39,
4156.
over
a wavelength range of 350±200 nm. The racemic
compound 9 lacked optical activity, while 10 and 11 exhibited
optical activity, with measured values of [y]226=+3470,
[y]245= 1248 and [y]226= 2961, [y]245=+1006, respectively.
10: mp 163.7±164.7 ꢀC; CD [y]226 +3470, [y]245 1248; 1H
NMR (200 MHz, Me2SO-d6) d 1.35 (d, 3H, J=7.1 Hz, CH3),
4.33 (m, 1H, CH), 6.69 (d, 1H, J=7.1 Hz, 2ꢀ amine NH), 6.97
(br s, 1H, NHamide), 7.25 (m, 1H, H-40), 7.35 (br m, 3H, NHa-
mide, NH2), 7.48 (m, 2H, H-30, H-50), 8.10 (s, 1H, H-3), 8.29 (d,
2H, J=8.0 Hz, H-20, H-60); 13C NMR (50 MHz, Me2SO-d6) d
18.5 (CH3), 50.5 (CH), 96.4 (C-3a), 119.5 (C-20, C-60), 124.9
(C-40), 128.9 (C-30, C-50), 134.2 (C3), 139.7 (C-10), 155.9 (C-
7a), 158.2 (C-4), 161.2 (C-6), 175.7 (CˆO); IR (KBr disc) 3397
7. Quinn, R. J.; Scammels, P. J. Tetrahedron Lett. 1991, 46,
6787.
8. 4 (Yield 73%); mp >350 ꢀC ; 1H NMR (200 MHz, Me2SO-
d6) d 7.27±8.15 (m, 5H, H-40, H-30, H-50, H-20, H-60), 7.42 (br s,
(1ꢀ amine NH); 3341, 3186 (1ꢀ amide NH); 1669 cm (CˆO);
1
HRMS (EI) 298.1425. Calcd for C14H15N7O.H+: 298.1416.
(EI) 298.1425. calcd for C14H15N7O.H+: 298.1416.