Pyrazolo[3,4-d]pyrimidine ribonucleosides related to 2-aminoadenosine and isoguanosine: Synthesis, deamination and tautomerism

Article abstract of DOI:10.1039/b708736e

The syntheses and properties of 8-aza-7-deazapurine (pyrazolo[3,4-d] pyrimidine) ribonucleosides related to 2-aminoadenosine and isoguanosine are described. Glycosylation of 8-aza-7-deazapurine-2,6-diamine 5 with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofur

Full text of DOI:10.1039/b708736e

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Pyrazolo[3,4-d]pyrimidine ribonucleosides related to 2-aminoadenosine and
isoguanosine: synthesis, deamination and tautomerism†
Frank Seela* and Kuiying Xu
Received 8th June 2007, Accepted 24th July 2007
First published as an Advance Article on the web 14th August 2007
DOI: 10.1039/b708736e
The syntheses and properties of 8-aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine) ribonucleosides
related to 2-aminoadenosine and isoguanosine are described. Glycosylation of 8-aza-7-
deazapurine-2,6-diamine 5 with 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (12) in the presence of
BF3·Et2O as a catalyst gave the N8 isomer 14 (73%) with a trace amount of the N9 isomer 13a (4.8%).
Under the same reaction conditions, the 7-halogenated 8-aza-7-deazapurine-2,6-diamines 6–8 afforded
the thermodynamically more stable N9 nucleosides 13b–d as the only products (53–70%). Thus, a
halogen in position 7 shifts the glycosylation from N8 to N9. The 8-aza-7-deazapurine-4,6-diamine
ribonucleosides 1a–d were converted to the isoguanosine derivatives 3a–d by diazotization of the
2-amino group. Although compounds 1a,b do not contain a nitrogen at position 7 (the enzyme binding
site), they were deaminated by adenosine deaminase; however, their deamination occurred with a much
slower velocity than that of the related purines. The pKa values indicate that the 7-non-functionalized
nucleosides 1a (pKa 5.8) and 15 (pKa 6.4) are possibly protonated in neutral conditions when
incorporated into RNA. The nucleosides 3a–d exist predominantly in the keto (lactam) form with KTAUT
(keto/enol) values of 400–1200 compared to 103–104 for pyrrolo[2,3-d]pyrimidine isoguanosine
derivatives 4a–c and 10 for isoguanosine itself, which will reduce RNA mispairing with U.
2ꢀ-deoxyribonucleotide duplexes increases significantly when 7-
Introduction
halogenated 8-aza-7-deazapurines replace purines.27 It has been
8-Aza-7-deazapurines (pyrazolo[3,4-d]pyrimidines) exhibit ex-
shown that the 7-halogenated 8-aza-7-deazapurine-2,6-diamine 2e
traordinary physical and biological properties1–4 if they are con-
forms a base pair with thymine that is as stable as a dG–dC base
pair,28 while the base pair of thymine and the non-functionalized
8-aza-7-deazapurine-2,6-diamine is not stronger than the dA–dT
pair.
stituents of nucleosides or oligonucleotides. 8-Aza-7-deazapurines
(purine numbering is used in the general discussion) closely
resemble the structure of purines and make their nucleosides
ideal mimics of the canonical purine constituents of DNA or
RNA. Since there is an absence of de novo purine biosynthesis
in most parasites, these organisms are wholly dependent in the
salvage pathway for the purine nucleoside metabolism. However,
they will accept certain 8-aza-7-deazapurines in place of purines.
For example, allopurinol (pyrazolo[3,4-d]pyrimidin-4(5H)-one),
which is an approved drug for the treatment of gout, was also
shown to be active against several leishmania and trypanosome
species.5–7 In both species allopurinol is converted to allopurinol
ribonucleoside 5ꢀ-phosphate.8,9 Monomeric 8-aza-7-deazapurine
nucleosides develop antiparasitic, antitumour and antiviral
activity.10–23 In particular, 8-aza-7-deazapurine-2,6-diamine and
its riboside 1a are powerful purine nucleoside antagonists.24,25 In
the form of their triphosphates these nucleosides are substrates
of polymerases, thereby making significant contributions to
DNA and RNA diagnostics.26 The thermal stability of oligo-
Recently, it was shown that 2-amino-7-deaza-2ꢀ-deoxyadenosine
(2a), the pyrrolo[2,3-d]pyrimidine analogue of 1a, is protonated
even under neutral conditions when it is a constituent of an
oligonucleotide, thereby forming a stable mismatched base pair
with dC. However, the halogenated derivatives 2b–d with lower
pKa values do not pair with dC.29 Base pairing ambiguity is also
found for other nucleosides that generate sufficient amounts of rare
tautomers such as 2ꢀ-deoxyisoguanosine, thereby forming strong
base pairs not only with dC or isoCd, but also stable mismatches
with dT and dG. This pairing ambiguity is significantly diminished
if the 7-deazapurines bear electron-withdrawing substituents in the
7-position.30
This manuscript investigates 7-halogenated 8-aza-7-deaza-
purine nucleosides related to 2-aminoadenosine and isoguanosine.
The 8-aza-7-deazapurine-2,6-diamine ribonucleoside 1a and its
7-substituted derivatives 1b–d (Scheme 1) were synthesized. As
compounds 1a–d do not contain an N-7 nitrogen, they might not
be deaminated by adenosine deaminase, as was observed for the
pyrrolo[2,3-d]pyrimidine analogues 2a–d. Chemical deamination
of 1a–d afforded the isoguanosine derivatives 3a–d. As it has
been reported that the tautomeric enol formation of isoguanine
nucleosides results in base pairing with T (or U),30 the ability
to form rare tautomers was studied on compounds 3a–d and
compared with the data obtained for purine and 7-deazapurine
nucleosides (4a–c).
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 48149 Mu¨nster, Germany and Labo-
ratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie,
Universita¨t Osnabru¨ck, Barbarastraße, 7, 49069, Osnabru¨ck, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; Web:
www.seela.net; Fax: +49 (0)251 53 406 857; Tel: +49 (0)251 53 406 500
† Electronic supplementary information (ESI) available: pKa measure-
ments, relative initial velocities of the deamination reaction, and deter-
mination of keto/enol populations. See DOI: 10.1039/b708736e
3034 | Org. Biomol. Chem., 2007, 5, 3034–3045
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Scheme 1 Structures of the nucleosides.
The low solubility of the base 5 in DMF or dichloromethane
resulted in low halogenation yields (32–65%). Moreover, difficul-
ties were encountered during the purification of 6 and 8 due to
poor solubility. Thus, for large scale preparation an alternative
route was used. Compound 9 bearing a hydrophobic isopropoxy
group was employed as a starting material. The halogenated
derivative 1135 was synthesized from 936 with NIS under reflux
in 1,2-dichloroethane. The former was converted to the diamino
compound 8 (74%) in aqueous ammonia in an autoclave. In
the same way, the chloro derivatives 10 and 6 were prepared
(Scheme 3).
Results and discussion
1. Synthesis of 8-aza-7-deazapurine ribonucleosides
The literature reports various synthetic routes to 8-aza-7-
deazapurine ribonucleosides:
(i) Davoll described the syntheses of the 8-aza-7-deazapurine-
2,6-diamine ribonucleosides 1a and 15 using the chloromercury
derivatives of the nucleobases and tri-O-benzoyl-D-ribofuranosyl
chloride as the sugar component.25 He obtained a mixture of N8
and N9 regioisomers in only 5–10% total yield.
(ii) The glycosylation of 2,6-substituted 8-aza-7-deazapurines
via the corresponding trimethylsilyl intermediates with tetra-O-
acetyl-b-D-ribofuranose and trimethylsilyl triflate as catalyst.31
(iii) Cottam et al.10,23 used BF3·Et2O as the catalyst under reflux
conditions, yielding various 8-aza-7-deazapurine ribonucleosides.
(iv) Recently, Bookser and Raffaele32 investigated the direct
glycosylation of 8-aza-7-deazapurine with microwave assistance.
Our laboratory has studied the synthesis of 2,6,7-trisubstituted
8-aza-7-deazapurine ribonucleosides using the corresponding 7-
halogenated 8-aza-7-deazapurines as precursors.33 As an efficient
route for the synthesis of 7-halogenated 8-aza-7-deazapurine
derivatives related to 2-aminoadenosine and isoguanosine does
not exist, this manuscript investigates the synthesis of a series of
7-substituted 8-aza-7-deazapurine derivatives using commercially
available 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (12) for
glycosylation. The starting material for the halogenated bases is
8-aza-7-deazapurine-4,6-diamine (5) prepared as described earlier
from guanidine carbonate and 3-amino-4-cyanopyrazole in a ring-
forming reaction.25 Compound 5 was halogenated with bromine
(→ 7)34 or halosuccinimides (NXS, X = Cl, I), affording the 7-
halogenated nucleobases 6–8 (Scheme 2).
Scheme 3 Reagents and conditions: (i) 10: NCS, ClCH2CH2Cl, 5 h, reflux;
11: NIS, ClCH2CH2Cl, 2.5 h, reflux; (ii) 25% NH4OH–dioxane (2 : 1),
120 ◦C, 24 h.
Next, the glycosylation of the nucleobases 5–8 was investigated.
First, we tried to perform the glycosylation with the sugar moiety
12 in MeCN with HMDS (base silylation) and tin chloride as
catalyst. This resulted in a complicated reaction mixture and very
little of the desired product. Later on we applied the procedure of
Cottam et al.,10,23 with BF3·Et2O as catalyst under reflux, which
gave satisfactory yields. According to an earlier observation,25
two isomers are expected with N8 and N9 as the glycosylation
sites, as was always found in the case of 2ꢀ-deoxyribonucleosides.35
However, in the case of the 7-halogenated 8-aza-7-deazapurine-
2,6-diamines 6–8, only the N9 isomers 13b–d (53–70%) (Scheme 4)
were obtained. In constrast, the glycosylation of the 7-non-
functionalized compound 5 resulted in two products, which were
assigned to be 13a (N9 4.8%) and 14 (N8 73%). Therefore the
7-halogen substituent has a significant influence on the outcome
of the glycosylation. The 7-non-functionalized compound 5 gave
a higher total glycosylation yield (78%) than the halogenated
bases 6–8 (53–70%), with the N8 isomer in excess. The 7-
halogen shifts the glycosylation site completely to N9, while
compound 5 (which is not hindered at the N8 position by a 7-
halogen substituent) can be more easily attacked at N8. Also, the
Scheme 2 Reagents and conditions: (i) 6: N-chlorosuccinimide (NCS),
DMF, 24 h, r.t.; 8: N-iodosuccinimide (NIS), CH2Cl2, 3 d, reflux, (ii) 7:
Br2, H2O, r.t., 1 h, reflux, 1 h.
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Scheme 4 Reagents and conditions: (i) BF3·Et2O, CH3NO2, 30 min, reflux; (ii) 0.5 M NaOCH3/CH3OH, overnight, r.t.
electron-withdrawing character of the halogen substituents oper-
ates in the same direction. As compound 5 reacts much faster
with the sugar (5 min, TLC monitoring) than compounds 6–8
(20–30 min) the formation of 14 is kinetically controlled, while
the N9 isomer is the thermodynamically more stable product. The
protected nucleosides 13a–d and 14 were deblocked with sodium
methoxide to give the free ribonucleosides 1a–d and 15 (76–92%)
(Scheme 4).
To access a higher yield of the N9 isomer 1a, the 2-amino-6-
isopropoxy-8-aza-7-deazapurine 936 (Scheme 5) was employed in
another glycosylation protocol. Compound 9 was first silylated
with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and then reacted
with the sugar 12 in the presence of TMSOTf (trimethylsilyl
trifluoromethanesulfonate) for 3 h at room temperature, affording
compound 16 in 44% yield (Scheme 5). Here, the N8 isomer 17
was isolated in only 15% yield. The moderate total glycosylation
yield resulted from a partial cleavage of the isopropoxy group
under the reaction conditions (HMDS, TMSOTf), which was
confirmed by a nucleoside side-product isolated from the reaction
mixture. 1H NMR spectra showed the absence of the isopropoxy
group, and a longer reaction time led to an increasing yield of
that compound, which after deprotection with sodium methoxide
(0.2 M) yielded 8-aza-7-deazaguanosine. Here the N9 isomer is
favoured from both the bulkiness and the electron-withdrawing
effect of the isopropoxy group. Compounds 16 and 17 were
converted to the nucleosides 1a and 15 by heating in aqueous
ammonia (Scheme 5). Next, compounds 1a–d were deaminated
selectively by diazotization at the 2-NH2 group, affording the
isoguanosine analogue 3a and its halogenated derivatives 3b–d
in yields of 42–57% (Scheme 6).
Scheme 6 Reagents and conditions: (i) NaNO2, AcOH, H2O, 50 min,
50 ◦C.
2. Deamination with adenosine deaminase
Selective deamination of the 6-NH2 group was expected by
the action of adenosine deaminase (ADA). ADA catalyzes the
irreversible hydrolysis of adenosine or 2ꢀ-deoxyadenosine to the
corresponding inosine and ammonia. Enzymatic deamination
by ADA has found many applications in nucleoside chemistry
and has led to functionalized purine nucleoside analogues in
drug discovery and medicinal chemistry. This reaction can be
performed on a preparative scale.37–41 As the ADA deaminates b-D
isomers exclusively, anomeric mixtures can be also separated.42
Considerable effort has been directed towards studies of the
structural requirements for adenosine derivatives to act as sub-
strates. When this method was applied to pyrrolo[2,3-d]pyrimidine
nucleosides, e.g. tubercidin (7-deazaadenosine), it was shown that
7-deazapurines are not deaminated (Table 1). It was therefore
Scheme 5 Reagents and conditions: (i) HMDS, (NH4)2SO4, 2 h, reflux; (ii) 1-O-acetyl-2,3,5-tri-O-benzyl-D-ribofuranose, TMSOTf, 3 h, r.t.; (iii) 28%
NH4OH–dioxane (4 : 1), overnight, 100 ◦C.
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Table 1 Relative initial velocities for adenosine derivatives deaminated with adenosine deaminasea
Compound
Rel. Vmax
Compound
Rel. Vmax
1a
0.27 ( 0.02)
0.18 ( 0.02)
0
0
Adenosine44
100
25
217
0
1b
2-Aminoadenosine44
1c
1d
8-Azaadenosine44
7-Deazaadenosine44
8-Aza-7-deazaadenosine
1.32 ( 0.01)
8-Aza-7-chloro-7-deaza-2ꢀ-deoxyadenosine
0
a For conditions see Experimental section and Supplementary information†.
concluded that N-7 is an essential enzyme binding site that
is necessary for the deamination reaction. This was supported
by the observation that another modified nucleoside, namely 8-
azaadenosine, is quickly deaminated (Table 1). As it is known
that 8-aza-7-deaza-2ꢀ-deoxyadenosine (which does not contain
a nitrogen at the 7-position) can be deaminated by adenosine
deaminase, it was concluded that this nitrogen is not essential
for deamination.43 As nothing was known about the substrate
properties of the diamino nucleoside 1a or the halogenated
derivatives 1b–d, the conversion to the corresponding 8-aza-7-
deazaguanosine by ADA was studied.
The relative initial velocity of the deamination was measured
UV-spectrophotometrically. The enzyme was diluted to a con-
centration (0.004 units ll−1) that warrants substrate saturation,
thereby making the initial velocity concentration-independent.
The initial velocities of compounds 1a,b and 2-aminoadenosine
were measured at 250 nm, and the data are compiled in Table 1.
It can be seen that the non-functionalized compound 1a and
the chloro compound 1b were deaminated, but with much
lower velocities than adenosine, while the deamination of 1c
and 1d does not occur. Consequently, N-7 is not essential for
the adenosine deaminase; however, the positional change of N-
7 (purine) to N-8 (pyrazolo[3,4-d]pyrimidine) leads to a 100-
fold decrease of the reaction rate (25 for 2-aminoadenosine
and 0.27 for 1a). This trend is also observed in the series of
adenosine derivatives (100 for adenosine and 1.32 for 8-aza-7-
deazaadenosine). A 7-chloro substituent introduced in compound
1a decreases the rate slightly (0.18 for 1b vs. 0.27 for 1a),
while the 7-chlorinated 8-aza-7-deazaadenosine is not deaminated
under the same conditions. Although compounds 1a and 1b
are deaminated rather slowly, the reaction can be performed
on preparative scale when a large excess of the enzyme is
employed.
3. Preparation and assignment of fixed tautomers of
8-aza-7-deazaisoguanine nucleosides
As the enol content of isoguanosine and corresponding derivatives
controls mismatch formation, the tautomeric keto–enol equilib-
rium of the isoguanosine derivatives 3a–d was determined (see
the following paragraph). For this it is necessary to correlate
the UV spectra of the particular tautomers to the structures of
the keto and enol forms. Consequently, the tautomeric species
were fixed by replacing the corresponding protons by methyl
groups. This will not influence UV spectra significantly, but will
allow us to make this assignment. Compound 3c was chosen as
an example. Four possible methylated tautomers are shown in
Scheme 7. As diazomethane directs the methylation to the oxo-
group,30 the fixed enol compound 3ce (Scheme 7) will be accessible.
However, two products were obtained when the reaction was
performed in methanol with diazomethane freshly prepared from
nitrosomethylurea (50% KOH in ether). According to the signals
1
of a methoxy group identified by H NMR and 13C NMR, and
a UV maximum at 264 nm (Fig. 1), one of the isomers was 3ce
(Scheme 7).
The other methylation product shows NMR signals of an
N-methyl group and identical UV spectra in both water and
dioxane (Fig. 1, compound 18), which indicate a fixed keto form –
(1H)keto, (3H)keto, or (8H)keto (Scheme 7). However, compound
18 is different from compound 3ck (fixed (1H)keto form). Also,
the N-8 methylated compound is excluded because there is no
chemical shift change for C-7 in the 13C NMR spectra (18 vs. 3c,
Table 3), which would be an indication of N-8 methylation.
Moreover, in the 13C NMR spectra of compound 18, C-4 has
a ∼10 ppm up-field shift compared to 3ce and 3ck. Therefore the
methyl group has to be attached to N-3 (compound 18, Scheme 7).
When compound 3c was treated with trimethylphosphate under
Scheme 7 Tautomers of compound 3c fixed by methylation.
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Fig. 1 UV spectra of compounds 3a–d, 3ck, 3ce and 18 in water (pH ∼6) (a) and in dioxane (containing 2% water) (b).
alkaline conditions, the fixed (1H)keto compound 3ck (Scheme 7)
was obtained together with compound 3ce. Compound 3ck carries
a methyl group as shown by the NMR spectra, and represents a
fixed keto form according to the UV absorption both in water and
in dioxane (Fig. 1), resembling the fixed keto form of the pyrrolo
derivative.30 Moreover, compound 3ck shows a 6 ppm up-field shift
for C2 compared to compound 3c (Table 3).
Fig. 1 shows the compilation of UV spectra of compounds 3a–d,
3ck, 3ce and 18. Compounds 3b–d are derivatives of 3a and show
similar UV spectra (see Fig. 1 and Table 2). The UV spectra of the
fixed tautomers 3ck and 3ce can be used to identify the fixed keto
and fixed enol forms for the whole series of nucleosides 3a–d.
5. Determination of pKa values and the keto/enol content of
nucleosides
Protonation and keto–enol tautomerism of nucleosides influence
base pairing selectivity and mismatch formation of DNA and
RNA. Thus, it is of importance to determine these physico-
chemical parameters in solution. At first, the pKa values of
the ribonucleosides 1a–d, 3a–d and 15 were determined by
spectrophotometric titration48 (pH 1.2–13.5) at 220–350 nm (see
Supplementary data†). As shown in Table 4, the pKa value of the 7-
non-functionalized nucleoside 1a is about 2 units higher than those
of the 7-halogenated compounds 1b–d. Therefore, compound 1a
is easier to protonate than the derivatives carrying an electron-
withdrawing halogen at the 7-position. The N8 nucleoside 15 shows
a pKa value (6.4) which is higher than that of the N9 compound 1a
(5.8). The table includes also the pKa values of the corresponding
purine and pyrrolo[2,3-d]pyrimidine nucleosides. The diamino
compounds 1a and 2a show almost no difference (5.8 for 1a and
5.7 for 2a), while that of the 2-aminoadenosine is significantly
lower (4.4). However, the halogenation has different effects on
pyrazolo[3,4-d]pyrimidines and pyrrolo[2,3-d]pyrimidines. In the
former series (1a–d), 7-halogenation decreases the pKa from 5.8
(1a) to about 3.7 (1b–d), but in the latter case only from 5.7 (2a)
to 4.7 (2b–d). In the series of the isoguanosine derivatives 3a, 4a
and isoG, the pyrazolo analogue 3a shows similar behaviour to
the purine congener isoG. The pyrrolo isoguanosine derivative 4a
is more basic (pKa 4.6) than the purine and pyrazolo analogues
(3.4 and 3.7 respectively). It is also shown that 7-halogenation
decreases the pKa value of both protonation and deprotonation
by about 1 unit in both the pyrrolo and pyrazolo derivatives (3a–d,
4a–c). Furthermore, it indicates that there is not much difference
between the various halogen substituents on the basicity of the
nucleosides in certain series of compounds. Within the oligonu-
cleotide chain the pKa values of protonation can be significantly
higher than those of the free nucleosides. Thus, the nucleosides 1a
and 15 might be protonated even under neutral conditions if they
are constituents of nucleic acids. The base pairing properties of the
2ꢀ-deoxyribo derivatives of 1a and 15 have already been studied;27,49
however, the pH-dependent character needs further investigation.
Next, the tautomeric equilibria of the isoguanine derivatives
3a–d were investigated and compared with corresponding purine
4. Spectroscopic data
Table 3 compiles the 13C NMR chemical shifts of all new
compounds. The assignments of the 13C NMR chemical data of
the base residues are made according to the literature,28,45,46 as are
the assignments of the carbon resonances of the sugar residues.47a
The table shows that compared to the 7-non-functionalized 8-aza-
7-deazapurines (5, 1a, 3a, 13a), the C-7 signals of the 7-chloro
derivatives (6, 1b, 3b, 13b) are shifted up-field (1–5 ppm). The shift
for the 7-bromo derivatives (7, 1c, 3c, 13c) is in the same direction
but is stonger (13–15 ppm), while for the iodo compounds (8,
1d, 3d, 13d) the up-field shift is the strongest (35–40 ppm). The
anomeric configuration was assigned to be b-D in view of the small
coupling constant (3JHH) for the anomeric proton47b (2–3 Hz for
13a–d, see Experimental section) as well as the chemical shifts of
C(1ꢀ), C(2ꢀ), and C(4ꢀ), which indicate the b-D anomers.47c
Table 2 UV absorption maxima of nucleosides in water and dioxane
Compound
kmax/nm (water)
kmax/nm (dioxane)a
3a
282, 251
286, 252
287, 249
289
264
287
262
265
266
268
264
300
270, 251
3b
3c
3d
3ce
3ck
18
268, 252
a 2% water was added for solubility.
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Table 3 13C NMR chemical shifts (ppm) of pyrazolo[3,4-d]pyrimidines and related ribonucleosides 1–18a
Compound
C-2 (C-6)b
C-4 (C-7a)b
C-5 (C-3a)
C-6 (C-4)b
C-7 (C-3)
C-1ꢀ
C-2ꢀ
C-3ꢀ
C-4ꢀ
C-5ꢀ
5
162.5
163.0
162.8
162.1
162.1
162.4
162.6
162.6
163.0
162.8
162.3
156.8
157.0
158.6
156.3
165.3
152.8
163.0
163.3
162.5
162.5
162.4
162.7
162.6
161.9
158.6
158.2
158.5
158.4
157.8
159.1
159.4
159.1
158.3
158.3
158.1
94.8
91.6
93.8
89.5
95.8
93.7
89.6
95.4
92.2
94.4
91.6
92.3
89.6
92.1
92.8
97.0
90.4
95.3
92.2
94.5
93.5
98.2
97.6
96.4
98.5
95.1
157.8
157.3
157.4
157.6
162.9
162.6
162.6
157.2
157.3
157.4
157.5
154.5
154.1
155.0
154.4
158.5
153.6
157.5
157.3
157.2
157.7
159.6
159.5
163.0
164.3
157.3
132.6
130.9
118.1
97.5
131.7
131.0
99.3
133.4
128.6
119.4
98.3
134.9
133.2
120.7
95.2
119.8
120.0
134.8
133.6
121.2
98.3
126.4
124.5
133.5
125.8
120.6
6
7
8
935
10
1135
1a
1b
1c
87.8
87.1
87.1
87.4
88.8
87.2
88.5
88.2
88.1
87.7
85.3
85.0
85.1
85.1
91.3
94.4
85.5
91.5
90.7
72.9
72.5
72.6
72.6
73.1
72.7
73.0
72.7
72.9
72.7
74.0
73.6
73.5
73.5
75.0
75.0
74.0
75.0
73.2
70.9
70.7
70.6
70.7
71.1
70.7
71.1
70.7
70.8
70.8
71.4
71.1
71.1
71.2
71.3
70.7
71.3
71.2
70.2
84.7
84.7
84.7
84.8
85.2
85.1
85.5
85.2
85.3
85.1
78.4
78.7
78.8
78.8
79.0
85.6
78.5
79.1
85.5
62.6
62.3
62.3
62.4
62.6
62.3
62.8
62.4
62.4
62.5
63.6
63.3
63.4
63.5
63.5
62.2
63.5
63.4
61.7
1d
3a
3b
3c
157.7
c
c
c
c
3d
3ce
3ck
13a
13b
13c
13d
14
15
16
17
18
157.5
156.6
158.3
158.4
158.1
157.8
162.2
162.5
158.5
164.0
148.9
a Measured in DMSO-d6 at 298 K. Carbons are labelled according to purine numbering; the labels in parentheses are the systematic numbers. b Tentative.
c Not detected.
Table 4 pKa values of nucleosidesa
Compound
Wavelengthb/nm
pKa
Compound
Wavelengthb/nm
pKa
1a
1b
1c
1d
3a
3b
3c
3d
15
257
275
276
276
265 (276)
295 (276)
294 (275)
266 (278)
305
5.8
3.7
3.6
3.7
3.7 (9.7)
2.7 (8.7)
2.7 (8.6)
2.7 (8.7)
6.4
2a29
285
285
286
288
255 (275)
264 (264)
264 (264)
296
5.7
4.9
4.8
4.9
4.6 (10.5)
3.9 (9.7)
3.8 (9.8)
4.4
2b
2c
2d
4a50
4b50
4c50
2-Aminoadenosine
isoG
250 (265)
3.4 (9.5)
a Measured in phosphate buffer (7.8 g NaH2PO4·H2O in 500 ml H2O) from pH 1.2 to pH 13.5. Values refer to protonation; those in parentheses refer to
deprotonation. b Wavelength with the most significant absorbance change.
and 7-deazapurine congeners. In aqueous solution, the canonical
nucleic acid constituents guanosine and 2ꢀ-deoxyguanosine exist
predominantly in the keto (lactam) form (KTAUT ≈ 104–105),51
which leads to an almost perfect Watson–Crick base pairing. As
only one of 104–105 of the Gd molecules is enolized, mispairing
is rare. Also, it was reported that iGd favours the keto (N–H)
form in aqueous solution and forms a stable base pair with iCd
(Scheme 8). However, the enol tautomer is present to a significant
amount (10%).52 This allows the formation of stable mismatches
with dT or dU (Scheme 8) and decreases the discrimination ability
Scheme 8 Keto and enol tautomers of isoguanine (and modified deriva-
tives) pair with iso C (or C) and T (or U).
of isoguanine during base pairing,53 which limits the use of the iGd
in hybridization. Since the keto–enol equilibrium of iGd depends
on the polarity of its microenvironment, one can expect that
the modification of nucleobases would influence this equilibrium,
resulting in alteration of the coding properties. It has already been
shown with pyrrolo[2,3-d]pyrimidine analogues of isoGd that the
enol content is only about 1/1000,54 and significantly decreased
when a 7-halogenated 7-deazaisoguanine replaces isoguanine.30
As a mimic of isoguanosine, compounds 3a–d might also prefer
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Scheme 9 Tautomeric equilibrium of nucleosides 3a–d.
the keto form in the aqueous solution. To prove our hypothesis,
the tautomeric equilibria between the keto and the enol forms of
3a–d were determined UV-spectrophotometrically in aqueous and
non-aqueous solutions.
As shown in Fig. 1 and Table 2, the UV spectra of compounds
3a–d, measured in water, showed the same maxima (250 nm and
286 nm), resembling the fixed keto form 3ck (Scheme 9). However,
under non-aqueous conditions (dioxane with 2% of water for
solubilization), the UV absorbance changes totally, showing the
maxima of the fixed enol form 3ce (264 nm). Therefore, it is
reasonable to assume that in aqueous solution the compounds 3a–
d exist almost entirely in the keto form, while in dioxane solution
the enol form is the predominant species.
Next, the tautomeric equilibria (KTAUT = [keto]/[enol]) of
compounds 3a–d were determined in dioxane–water mixtures
ranging from 98 : 2 to 5 : 95. The ratio of the tautomers
was determined by the multiwavelength method of Dewar and
Urch.55 Data were collected at around 265 and 286 nm, where the
extinction coefficients of both forms show the highest difference.
When the water–dioxane ratio increases, the UV spectra for
compound 3a–d shifted gradually, more and more resembling the
UV spectra of the keto form. When the water content was higher
than 55%, the spectra became solvent-independent and the band
at 265 nm nearly disappeared, while the absorbance at 286 nm
increased to the level of the pure keto form. By adding more water,
the whole spectrum is moved to shorter wavelength due to the
increase of the polarity of the solvent system (see Supplementary
data†). The composition of keto and enol forms was maintained at
the same level. From these data, the KTAUT values were determined
quantitatively by the method of Shugar et al.52 and Voegel et al.56
A plot of the logarithm of the tautomeric equilibrium constant
for 3a–d versus the polarity parameter ET(30)57–59 of a set of the
dioxane–water mixtures is shown in Fig. 2. When ET(30) is in the
range 38–50, the relationship between log[KTAUT ] and ET(30) is
linear. This can be used to estimate the value of KTAUT in aqueous
solution. By extrapolating this linear relationship to the ET(30)
value of water (63.1),59 the tautomeric equilibrium constants for
compound 3a–d were calculated as 400 for 3a, 750 for 3b, 550
for 3c and 1200 for 3d. These values are smaller than those of
the corresponding pyrrolo[2,3-d]pyrimidine analogues 4a–d (103–
104)30,54 but larger than that of isoguanosine (10)52 (Table 5). This
means that the pyrazolo[3,4-d]pyrimidines 3a–d are more enolized
than the pyrrolo[2,3-d]pyrimidines 4a–d, while isoguanosine itself
shows the highest enol content. Enol formation of isoguanosine is
Table 5 The tautomerism equilibrium constants for isoguanosine
derivatives
Compound
KTAUT
Compound
KTAUT
3a
3b
3c
3d
400
750
550
4a30
4b30
4c30
1880
12 800
19 400
10
1200
iGd52
Fig. 2 Plot of log([enol]/[keto]) versus ET(30) for compound 3a (a) and 3c (b) in mixtures of dioxane (ET(30) = 36.0) and water (ET(30) = 63.1).
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thought to be the reason for mismatch formation, and therefore
the position exchange of N-7 and C-8 results in a decrease of the
mispairing of isoguanosine. Moreover, the halogenated derivatives
3b–d have almost the same ratio of enol content as 3a. Thus the
halogens should not change the base recognition of the 8-aza-
7-deazaisoguanosine derivative (3a) but enhance the base pair
stability even in mismatches, which is consistent with observation
in the case of oligonucleotides.46
S. The extinction coefficients of the substrate and the product at
certain wavelengths were measured as esub and epro; the difference
between them is defined as De = epro − esub. The relative Vmax of the
sample was calculated according to the equation
Vmax(sample) = Vmax(standard) × (SsampleDestandard/SstandardDesample).
2-Aminoadenosine was taken as standard substrate with a
relative Vmax of 25.44 For details see Supplementary data†.
Conclusions
3-Chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (6)
7-Halogenated 8-aza-7-deazapurine-2,6-diamines and the cor-
responding ribonucleosides (1a–d, 15) were synthesized. The
glycosylation of the 7-non-functionalized 8-aza-7-deazapurine-
2,6-diamine 5 yielded the N8 nucleoside (73%) as main product
with very little of the N9 compounds (4.8%). A 7-halogen
substituent shifts the glycosylation reaction exclusively to N9.
The resulting diamino compounds 1a–d were further converted
to the isoguanosine derivatives 3a–d. 8-Aza-7-deazapurine-2,6-
diamine ribonucleoside 1a and 7-chloro derivative 1b, which do
not contain a nitrogen in position 7, are substrates of ADA,
but with significantly lower relative Vmax values than the purine
congener 2-aminoadenosine, while the bromo and iodo derivatives
(1c and 1d) could not be deaminated with detectable velocities.
Compound 1a and 15 have pKa values as high as 5.8 and 6.4, which
indicates possible protonation in oligonucleotides under neutral
conditions, resulting in mismatch formation. For compounds
3a–d the keto–enol equilibrium constant KTAUT is between 400–
1200. Thus, it is expected that they should show better mismatch
discrimination than isoguanosine. As protonation of 15 occurs at
pH 6.4, protonation of oligonucleotides might occur under neutral
conditions. As a consequence, compounds 1a–d and 3a–d might
be used as pH-dependent molecular switches. The synthesis and
properties of the corresponding oligonucleotides is currently being
evaluated.
From 5. To a solution of 1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (5)26 (1.5 g, 10 mmol) in anhydrous DMF (50 cm3),
N-chlorosuccinimide (2.02 g, 15 mmol) was added. The mixture
was kept stirred at room temperature. After 24 h, the mixture
was evaporated to dryness. The remaining residue was adsorbed
on silica gel (15 g) and applied to a flash chromatography (FC)
silica gel column (10 × 6 cm, CH2Cl2–CH3OH, 20 : 1) to yield
a slightly yellow solid; recrystallization from MeOH afforded
yellowish crystals (0.59 g, 32%).
From 10. 6-Amino-3-chloro-4-isopropoxy-1H-pyrazolo[3,4-
d]pyrimidine (10) (100 mg, 0.44 mmol) was suspended in conc.
aq. NH3–dioxane (2 : 1, 30 cm3) and placed in an autoclave. The
suspension was stirred at 120 ◦C for 24 h. The clear solution
was evaporated until precipitation occurred. The precipitate was
filtrated and washed with acetone, affording compound 6 as
slightly yellow amorphous solid (66 mg, 81%) (Found: C, 32.60; H,
2.80; N, 45.21. C5H5ClN6 requires C, 32.53; H, 2.73; N, 45.53%);
mp > 300 ◦C (from MeOH); TLC (silica gel, CH2Cl2–CH3OH,
10.1): Rf 0.20; kmax(MeOH)/nm 275 (e/dm3 mol−1 cm−1 6900), 257
(7000) and 224 (29 200); dH(250 MHz; DMSO-d6; Me4Si) 6.19
(2 H, br s, NH2); 6.79 (2 H, br s, NH2), 12.61 (1 H, br s, NH).
Experimental
3-Iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (8)
General
From 5. To a suspension of 1H-pyrazolo[3,4-d]pyrimidine-
4,6-diamine (5)26 (0.75 g, 5 mmol) in anhydrous 1,2-dichloroethane
(200 cm3), N-iodosuccinimide (1.35 g, 6 mmol) was added. The
mixture was kept stirring under reflux for 3 d. The mixture was
cooled and evaporated to dryness and purified by FC (silica gel
column, 10 × 5 cm, CH2Cl2–CH3OH, 20 : 1) to yield a slightly
yellow solid (0.9 g, 65%).
All chemicals were purchased from Acros, Fluka, or Sigma-
Aldrich. TLC: aluminium sheets coated with silica gel 60 F254
(0.2 mm, VWR International). Flash chromatography (FC): 0.4
bar, silica gel 60 (Merk, Darmstadt, Germany). Mp: Linstro¨m ap-
paratus, not corrected. UV spectra: U-3200 UV-Vis spectrometer
(Hitachi, Japan). NMR spectra: Avance-250 or AMX-500 spec-
trometers (Bruker); d values in ppm relative to Me4Si as internal
standard. J values are in Hz. Elemental analyses: performed by
the Mikroanalytisches Laboratorium Beller, Go¨ttingen, Germany.
Adenosine deaminase (EC 3.5.4.4), Type V from bovine spleen.
Solution in 50% glycerol, 50 mM potassium phosphate, pH 6.0,
160 units per ml (Sigma). The adenosine deaminase was diluted
with 0.06 M Sørensen buffer (pH 7.0). A 40-fold diluted solution
(0.004 units per ll) was used. The substrate was dissolved in the
same buffer, after the deaminase (1 ll, 0.004 units) was added; the
UV absorbance was detected and recorded at certain wavelengths
where the UV spectra have the maximal changes. The absorption
at this wavelength vs. the time was plotted. The slope of the linear
relationship between the absorbance and the time was taken as
From
11. 6-Amino-3-iodo-4-isopropoxy-1H-pyrazolo[3,4-
d]pyrimidine (11)35 (5.5 g, 17.2 mmol) was suspended in 28%
NH3–dioxane (2 : 1, 350 cm3) and placed in a steel bomb. The
suspension was stirred at 120 ◦C for 24 h. The clear solution
was evaporated until precipitation occurred. The precipitate was
filtrated and washed with acetone, affording compound 8 as a
colourless amorphous solid (3.5 g, 74%) (Found: C, 21.68; H,
1.82; N, 30.26. C5H5IN6 requires C, 21.76; H, 1.83; N, 30.45%);
TLC (silica gel, CH2Cl2–CH3OH, 10.1): Rf 0.23; kmax(MeOH)/nm
271 (e/dm3 mol−1 cm−1 7800) and 224 (29 200); dH(250 MHz;
DMSO-d6; Me4Si) 6.17 (2 H, br s, NH2), 6.53 (2 H, br s, NH2),
12.85 (1 H, br s, NH).
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6-Amino-3-chloro-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidine
(10)
13.46. C31H25ClN6O7 requires C, 59.19; H, 4.01; N, 13.36%); TLC
(silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.49; kmax(MeOH)/nm
275 (e/dm3 mol−1 cm−1 12 300), 262 (12 200) and 227 (68 700);
dH(250 MHz; DMSO-d6; Me4Si) 4.53–4.65 (2 H, m, 2 × 5ꢀ-H),
4.82 (1 H, m, 4ꢀ-H), 6.07 (1 H, m, 3ꢀ-H), 6.19 (1 H, m, 2ꢀ-H), 6.43
(1 H, d, J 3.35, 1ꢀ-H), 6.53 (2 H, br s, NH2), 7.02 (2 H, br s, NH2),
7.46–8.07 (15 H, m, 3 × Ph).
Compound 936 (400 mg, 2.07 mmol) was suspended in 1,2-
dichloroethane (200 cm3), N-chlorosuccinimide (NCS) (300 mg,
2.25 mmol) was added, and the mixture was stirred under reflux
for 5 h. The solution was cooled, evaporated to dryness and the
residue purified by FC (silica gel column, 10 × 5 cm, elution
with CH2Cl2–EtOAc 2 : 1) to yield a colourless solid (200 mg,
42%). Recrystallization from methanol afforded colourless needles
(Found: C, 42.10; H, 4.36; N, 30.35. C8H10ClN5O requires C,
42.21; H, 4.43; N, 30.76%); mp 253 ◦C (From MeOH); TLC
(silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.44; kmax(MeOH)/nm
278 (e/dm3 mol−1 cm−1 7300), 248 (5700) and 224 (29 200);
dH(250 MHz; DMSO-d6; Me4Si) 1.35 (6 H, m, 2 × CH3), 5.46
(1 H, m, CH3CHCH3), 6.79 (2 H, br s, NH2), 12.92 (1 H, br s,
NH).
3-Bromo-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-1H-
pyrazolo[3,4-d]pyrimidine-4,6-diamine (13c)
As described for 13b, with 3-bromo-1H-pyrazolo[3,4-d]-
pyrimidine-4,6-diamine (7)34 (2.29 g, 10 mmol) and 12 (7.56 g,
15 mmol), 13c was obtained (4.69 g, 69.7%) as a colourless foam
(Found: C, 55.10; H, 3.82; N, 12.40. C31H25BrN6O7 requires C,
55.29; H, 3.74; N, 12.48%); TLC (silica gel, CH2Cl2–CH3OH, 20 :
1): Rf 0.33; kmax(MeOH)/nm 275 (e/dm3 mol−1 cm−1 12 000), 263
(12 000) and 229 (66 500); dH(250 MHz; DMSO-d6; Me4Si) 4.59
(2 H, m, 2 × 5ꢀ-H), 4.83 (1 H, m, 4ꢀ-H), 6.07 (1 H, m, 3ꢀ-H), 6.21
(1 H, m, 2ꢀ-H), 6.44 (1 H, d, J 3.55, 1ꢀ-H), 6.57 (4 H, br s, 2 ×
NH2), 7.44–8.08 (15 H, m, 3 × Ph).
1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-1H-pyrazolo[3,4-
d]pyrimidine-4,6-diamine (13a) and 2-(2,3,5-tri-O-benzoyl-b-
D-ribofuranosyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (14)
1H-Pyrazolo[3,4-d]pyrimidine-4,6-diamine
(5)26
(1.00
g,
3-Iodo-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-1H-
pyrazolo[3,4-d]pyrimidine-4,6-diamine (13d)
6.67 mmol) was suspended in nitromethane (20 cm3), and
1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (12, 5.04 g,
10 mmol) was added. The mixture was brought to reflux
As described for 13b, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (8) (552 mg, 2 mmol) and 12 (1.52 g, 3 mmol) gave 13d
(770 mg, 53.5%) as a colourless foam (Found: C, 51.95; H, 3.35; N,
11.82. C31H25IN6O7 requires C, 51.68; H, 3.50; N, 11.66%); TLC
(silica gel, CH2Cl2–CH3OH, 20 : 1): Rf 0.34; kmax(MeOH)/nm
275 (e/dm3 mol−1 cm−1 12 100), 264 (12 400) and 230 (67 600);
dH(250 MHz; DMSO-d6; Me4Si) 4.58 (2 H, m, 2 × 5ꢀ-H), 4.82
(1 H, m, 4ꢀ-H), 6.03 (1 H, t, J 5.6, 3ꢀ-H), 6.23 (1 H, m, 2ꢀ-H), 6.42
(1 H, d, J 3.83, 1ꢀ-H), 6.49 (2 H, br s, NH2), 6.75 (2H, br s, NH2),
7.43–8.07 (15 H, m, 3 × Ph).
◦
(130 C) and then BF3·OEt2 (2.12 cm3, 16.67 mmol) was added.
Immediately, the mixture became clear and then slowly became
dark. The mixture was kept stirred at this temperature for 15 min.
After being cooled to r.t. (ice bath), the reaction mixture was
evaporated, and the remaining residue was purified by FC (silica
gel column, 12 × 5 cm, CH2Cl2–CH3OH 50 : 1 → 20 : 1);
compound 13a was obtained (190 mg, 4.8%) as a colourless solid
from the faster migrating zone (Found: C, 62.65; H, 4.50; N,
14.10. C31H26N6O7 requires C, 62.62; H, 4.41; N, 14.13%); TLC
(silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.59; kmax(MeOH)/nm
275 (e/dm3 mol−1 cm−1 13 000), 258 (12 100) and 226 (59 500);
dH(250 MHz; DMSO-d6; Me4Si) 4.52–4.55 (1 H, m, 5ꢀ-H),
4.61–4.64 (1 H, m, 5ꢀ-H), 4.84 (1 H, m, 4ꢀ-H), 6.18–6.22 (2 H, m,
3ꢀ-H and 2ꢀ-H), 6.26 (2 H, s, NH2), 6.46 (1 H, d, J 2.5, 1ꢀ-H), 7.25
(2 H, br s, NH2); 7.45–8.03 (17 H, m, 3 × Ph and NH2), 8.35 (1 H,
s, 3-H).
1-(b-D-Ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(1a)
From
13a. 1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-1H-
pyrazolo[3,4-d]pyrimidine-4,6-diamine (13a) (183 mg, 0.31 mmol)
was suspended in 0.5 M NaOCH3/MeOH (5 cm3) and the mixture
was kept stirred at r.t. overnight. The mixture was evaporated to
dryness. The residue was adsorbed on a small amount of silica
gel (2 g) and loaded on the top of a FC silica gel column (5 × 2.5
cm), eluting with CH2Cl2–CH3OH (10 : 1 → 2 : 1), furnishing
compound 1a (80 mg, 92%) as a colourless solid.
Compound 14 was collected from the second zone as a colour-
less solid (2.9 g, 73%), recrystallization from CH2Cl2–CH3OH gave
pale red crystals (Found: C, 62.64; H, 4.35; N, 14.15. C31H26N6O7
requires C, 62.62; H, 4.41; N, 14.13%); mp 165 ◦C (from CH2Cl2
and MeOH); TLC (silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.42;
kmax(MeOH)/nm 297 (e/dm3 mol−1 cm−1 6400), 283 (7200), 266
(9300) and 227 (51 100); dH(250 MHz; DMSO-d6; Me4Si) 4.52–
4.67 (2 H, m, 2 × 5ꢀ-H), 4.89 (1 H, m, 4ꢀ-H), 6.10 (1 H, m, 3ꢀ-H),
6.24 (1 H, m, 2ꢀ-H) 6.37 (2 H, br s, NH2), 6.63 (1 H, s, 1ꢀ-H),
7.43–7.99 (17 H, m, 3 × Ph and NH2), 8.41 (1 H, s, 3-H).
From 16. Compound 16 (see below) (650 mg, 1.02 mmol) was
dissolved in 30 cm3 dioxane and then 120 cm3 aqueous ammonia
(28%) was added. The mixture was kept stirred in an autoclave at
◦
100 C overnight. The reaction mixture was evaporated and the
residue was adsorbed on silica gel (3 g) and applied to a silica gel
column (8 × 2.5 cm). Elution with CH2Cl2–CH3OH (20 : 1 → 5 :
1) gave compound 1a (200 mg, 69%) as a colourless solid (Found:
C, 42.40; H, 4.90; N, 29.90. C10H14N6O4 requires C, 42.55; H,
5.00; N, 29.77%); TLC (silica gel, CH2Cl2–CH3OH, 4 : 1): Rf 0.14;
kmax(MeOH)/nm 276 (e/dm3 mol−1 cm−1 8300), 259 (7800) and
224 (28 600); dH(250 MHz; DMSO-d6; Me4Si) 3.49–3.58 (2 H, m,
3-Chloro-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-1H-
pyrazolo[3,4-d]pyrimidine-4,6-diamine (13b)
As described for 13a, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-
4,6-diamine (6) (230 mg, 1.25 mmol) was converted to 13b.
Colourless foam (485 mg, 62%) (Found: C, 59.23; H, 3.96; N,
3042 | Org. Biomol. Chem., 2007, 5, 3034–3045
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2 × 5ꢀ-H), 3.83 (1 H, m, 4ꢀ-H), 4.24 (1 H, m, 3ꢀ-H), 4.48 (1 H, m,
2ꢀ-H), 4.88 (1 H, t, J 5.6, 5ꢀ-OH), 5.02 (1 H, d, J 5.2, 3ꢀ-OH), 5.26
(1 H, d, J 5.6, 2ꢀ-OH), 5.90 (1 H, d, J 4.5, 1ꢀ-H), 6.22 (2 H, br s,
NH2), 7.16 (2H, br s, NH2), 7.86 (1 H, s, 3-H).
3-Iodo-1-(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]-
pyrimidine-4,6-diamine (1d)
As described for 1a–c, from 3-iodo-1-(2,3,5-tri-O-benzoyl-b-D-
ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (13d)
(1.1 g, 1.53 mmol), compound 1d was obtained (474 mg, 76%)
as a colourless solid. Recrystallisation from MeOH–H2O af-
forded colourless needles (Found: C, 29.72; H, 3.32; N, 20.39.
2-(b-D-Ribofuranosyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(15)
◦
C10H13IN6O4 requires C, 29.43; H, 3.21; N, 20.59%); mp 248 C
1-(2,3,5-Tri-O-benzoyl-b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]-
pyrimidine-4,6-diamine (14) (180 mg, 0.30 mmol) was suspended
in 0.5 M NaOCH3/MeOH (5 cm3) and the mixture was kept
stirred at r.t. overnight. The solvent was evaporated and the
residue was dissolved in distilled water (30 cm3). The resulting
solution was applied to the top of a Serdolit AD-4 column
(20 × 4 cm) and the compound eluted with water. The fractions
containing the desired compound were combined and condensed
to furnish compound 15 (67 mg, 79%) as a colourless solid (Found:
C, 42.80; H, 4.93; N, 29.59. C10H14N6O4 requires C, 42.55; H,
5.00; N, 29.77%); kmax(MeOH)/nm 297 (e/dm3 mol−1 cm−1
6900), 265 (7600) and 225 (26 400); dH(250 MHz; DMSO-d6;
Me4Si) 3.46–3.58 (1 H, m, 5ꢀ-H), 3.64–3.67 (1 H, m, 5ꢀ-H),
3.97 (1 H, m, 4ꢀ-H), 4.13 (1 H, m, 3ꢀ-H), 4.35 (1 H, m, 2ꢀ-H),
5.02–5.53 (3 H, br m, 5ꢀ-OH, 3ꢀ-OH and 2ꢀ-OH), 5.70 (1 H, d,
J 3.6, 1ꢀH), 5.77 (2 H, br s, NH2), 7.21 (2 H, br s, NH2), 8.26
(1 H, s, 3-H).
(dec.) (from MeOH–H2O); TLC (silica gel, CH2Cl2–CH3OH, 5 :
1): Rf 0.33; kmax(MeOH)/nm 279 (e/dm3 mol−1 cm−1 8000), 263
(9000) and 230 (29 900); dH(250 MHz; DMSO-d6; Me4Si) 3.49–
3.63 (2 H, m, 2 × 5ꢀ-H), 3.89 (1 H, m, 4ꢀ-H), 4.17 (1 H, m, 3ꢀ-H),
4.54 (1 H, m, 2ꢀ-H), 4.68 (1 H, t, J 5.2, 5ꢀ-OH), 5.11 (1 H, d, J 5.0,
3ꢀ-OH), 5.36 (1 H, d, J 5.8, 2ꢀ-OH), 5.91 (1 H, d, J 4.8, 1ꢀ-H), 6.43
(2 H, br s, NH2), 6.72 (2 H, br s, NH2).
6-Amino-4-isopropoxy-1-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-
1H-pyrazolo[3,4-d]pyrimidine (16) and 6-amino-4-isopropoxy-2-
(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2H-
pyrazolo[3,4-d]pyrimidine (17)
A
suspension of 6-amino-4-isopropoxy-1H-pyrazolo[3,4-d]-
pyrimidine (9,36 386 mg, 2 mmol) and a catalytic amount of
(NH4)2SO4 in HMDS (10 cm3) was refluxed for 2 h. The excess of
HMDS was removed by evaporation and the residue was dissolved
in 1,2-dichloroethane (6 cm3). To this solution compound 12
(1.21 g, 2.4 mmol) was added followed by the addition of
trimethylsilyl trifluoromethanesulfonate (0.6 cm3, 5 mmol). The
mixture was kept stirred at r.t. After 3 h, the mixture was diluted
with CH2Cl2 (20 cm3) and washed with 5% aqueous NaHCO3. The
organic layer was dried and filtered. The filtrate was evaporated to
a dryness and dissolved in dichloromethane (4 ml) and applied
to the top of a FC silica gel column (10 × 2.5 cm). Elution
with CH2Cl2–CH3OH (100 : 1 → 20 : 1) yielded two compounds;
the compound eluting first was 16 (566 mg, 44%), obtained as a
colourless foam (Found: C, 64.00; H, 5.10; N, 10.74. C34H31N5O8
requires C, 64.00; H, 4.90; N, 10.98%); TLC (silica gel, CH2Cl2–
CH3OH, 20 : 1): Rf 0.66; kmax(MeOH)/nm 275 (e/dm3 mol−1 cm−1
11 800) and 255 (53 500); dH(250 MHz; DMSO-d6; Me4Si) 1.33
(3 H, s, CH3), 1.36 (3 H, s, CH3), 4.51–4.61 (3 H, m, 2 × 5ꢀ-H and
4ꢀ-H), 4.84 (1 H, m, 3ꢀ-H), 5.48 (1 H, m, CH3CHCH3), 6.16–6.21
(2 H, m, 2ꢀ-H and 3ꢀ-H), 6.51 (1 H, d, J 2.4, 1ꢀ-H), 6.93 (2 H, br s,
NH2), 7.42–8.01 (16 H, m, 3 × Ph and 3-H).
3-Chloro-1-(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]-
pyrimidine-4,6-diamine (1b)
As described for 1a (from 13a), starting from 3-chloro-1-(2,3,5-
tri-O-benzoyl-b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-
4,6-diamine (13b, 180 mg, 0.29 mmol), compound 1b (80 mg,
88%) was furnished as a colourless solid (Found: C, 37.91;
H, 4.27; N, 26.43. C10H13ClN6O4 requires C, 37.92; H, 4.14;
N, 26.54%); TLC (silica gel, CH2Cl2–CH3OH, 5 : 1): Rf 0.30;
kmax(MeOH)/nm 276 (e/dm3 mol−1 cm−1 8600), 261 (9300) and
227 (34 900); dH(250 MHz; DMSO-d6; Me4Si) 3.38–3.54 (2 H, m,
2 × 5ꢀ-H), 3.82 (1 H, m, 4ꢀ-H), 4.10 (1 H, m, 3ꢀ-H), 4.44 (1 H, m,
2ꢀ-H), 4.77 (1 H, t, J 5.5, 5ꢀ-OH), 5.06 (1 H, d, J 3.9, 3ꢀ-OH), 5.31
(1 H, d, J 5.6, 2ꢀ-OH), 5.86 (1 H, d, J 4.7, 1ꢀ-H), 6.44 (2 H, br s,
NH2), 6.97 (2 H, br s, NH2).
The compound eluting last was 17 (185 mg, 15%), obtained as a
pale yellow foam (Found: C, 63.92; H, 5.24; N, 10.88. C34H31N5O8
requires C, 64.00; H, 4.90; N, 10.98%); TLC (silica gel, CH2Cl2–
CH3OH, 20 : 1): Rf 0.27; kmax(MeOH)/nm 282 (e/dm3 mol−1 cm−1
7700) and 268 (8400); dH(250 MHz; DMSO-d6; Me4Si) 1.32–1.40
(6 H, m, 2 × CH3), 4.56–4.63 (2 H, m, 2 × 5ꢀ-H), 4.90 (1 H, m,
4ꢀ-H), 5.45–5.50 (1 H, m, CH3CHCH3), 6.12 (1 H, m, 3ꢀ-H), 6.27
(1 H, m, 2ꢀ-H), 6.50 (1 H, d, J 1.7, 1ꢀ-H), 6.56 (2 H, s, NH2),
7.43–8.02 (15 H, m, 3 × Ph), 8.49 (1 H, s, 3-H).
3-Bromo-1-(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]-
pyrimidine-4,6-diamine (1c)
As described for 1a,b, but starting from 3-bromo-1-(2,3,5-tri-O-
benzoyl-b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (13c, 1.59 g, 2.36 mmol), compound 1c was obtained
as a colourless solid (648 mg, 76%). Recrystallization from H2O
gave colourless crystals (Found: C, 33.66; H, 3.64; N, 23.67.
C10H13BrN6O4 requires C, 33.26; H, 3.63; N, 23.27%); mp 236 ◦C
(from H2O); TLC (silica gel, CH2Cl2–CH3OH, 5 : 1): Rf 0.30;
kmax(MeOH)/nm 278 (e/dm3 mol−1 cm−1 7300), 262 (7600) and
228 (29 400); dH(250 MHz; DMSO-d6; Me4Si) 3.50 (2 H, m, 2 ×
5ꢀ-H), 3.83 (1 H, m, 4ꢀ-H), 4.10 (1 H, m, 3ꢀ-H), 4.45 (1 H, m, 2ꢀ-H),
4.79 (1 H, m, 5ꢀ-OH), 5.07 (1 H, d, J 4.1, 3ꢀ-OH), 5.32 (1 H, d, J
4.8, 2ꢀ-OH), 5.87 (1 H, d, J 4.4, 1ꢀ-H), 6.42 (2 H, br s, NH2), 6.84
(2 H, br s, NH2).
4-Amino-1-(b-D-ribofuranosyl)-1H-pyrazolo-
[3,4-d]pyrimidine-6-one (3a)
To a stirred solution of NaNO2 (69 mg, 1 mmol) in H2O (2.9 cm3) at
50 ◦C, 1-(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-
diamine (1a) (70 mg, 0.25 mmol) was added followed by AcOH
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(0.16 cm3, 2.8 mmol) dropwise. The mixture became a clear
solution within 5 min. The reaction was kept stirred at 50 ◦C
and monitored by TLC; after 50 min, the mixture was diluted
with H2O (10 cm3) and concentrated aq. NH3 was added until
pH 8 was reached. The solution was applied to a column (20 ×
2 cm, Serdolit AD-4 resin). The column was washed with H2O
(200 cm3), and the product was eluted with H2O–iPrOH (95 : 1,
300 cm3), yielding 3a (30 mg, 43%) as a colourless solid (Found:
C, 42.34; H, 4.73; N, 24.80. C10H13N5O5 requires C, 42.40; H, 4.63;
N, 24.73%); kmax(MeOH)/nm 282 (e/dm3 mol−1 cm−1 5600) and
262 (7300); dH(250 MHz; DMSO-d6; Me4Si) 3.52–3.58 (2 H, m,
2 × 5ꢀ-H), 3.85 (1 H, m, 4ꢀ-H), 4.12 (1 H, m, 3ꢀ-H), 4.47 (1 H, m,
2ꢀ-H), 5.07–5.30 (3 H, m, 5ꢀ-OH, 3ꢀ-OH and 2ꢀ-OH), 5.80 (1 H, d,
J 4.5, 1ꢀ-H), 7.93 (1 H, s, 3-H), 8.67 (2 H, br s, NH2).
The mixture was kept stirred at r.t. for 20 min and then evaporated.
The residue was adsorbed to silica gel (1.5 g) and applied to the
top of a FC silica gel column (10 × 2.5 cm), elution with CH2Cl2–
CH3OH (50 : 1 → 5 : 1), affording compound 3ce (39 mg, 29%)
and 18 (30 mg, 22%).
Compound 3ce was recrystallized from MeOH–CH2Cl2, giv-
ing colourless crystals (Found: C, 35.36; H, 4.00; N, 18.51.
C11H14BrN5O5 requires C, 35.12; H, 3.75; N, 18.62%); mp 226 ◦C
(from MeOH and CH2Cl2); TLC (silica gel, CH2Cl2–CH3OH,
10 : 1): Rf 0.29; kmax(MeOH)/nm 263 (e/dm3 mol−1 cm−1 8300);
dH(250 MHz; DMSO-d6; Me4Si) 3.33–3.58 (2 H, m, 2 × 5ꢀ-H),
3.86 (4 H, m, 4ꢀ-H and OCH3), 4.13 (1 H, m, 3ꢀ-H), 4.54 (1 H, m,
2ꢀ-H), 4.77 (1 H, t, J 5.6, 5ꢀ-OH), 5.16 (1 H, d, J 5.2, 3ꢀ-OH), 5.39
(1 H, d, J 5.9, 2ꢀ-OH), 5.95 (1 H, d, J 4.8, 1ꢀ-H), 8.05 (2 H, br s,
NH2).
Compound 18 was recrystallized from MeOH, giving colourless
crystals (Found: C, 35.02; H, 4.12. C11H14BrN5O5 requires C,
35.12; H, 3.75); mp 205 ◦C (dec.) (from MeOH–CH2Cl2); TLC
(silica gel, CH2Cl2–CH3OH, 5 : 1): Rf 0.47; kmax(MeOH)/nm 269
(e/dm3 mol−1 cm−1 8500) and 252 (10 300); dH(250 MHz; DMSO-
d6; Me4Si) 3.27–3.54 (2 H, m, 2 × 5ꢀ-H), 3.61 (3 H, s, CH3), 3.90
(1 H, m, 4ꢀ-H), 4.18 (1 H, m, 3ꢀ-H), 4.70 (2 H, m, 2ꢀ-H and 5ꢀ-OH),
5.21 (1 H, d, J 5.9, 3ꢀ-OH), 5.43 (1 H, d, J 5.4, 2ꢀ-OH), 6.00 (1 H,
d, J 3.4, 1ꢀ-H), 6.56 (1 H, br s, NH of NH2), 7.97 (1 H, br s, NH
of NH2).
4-Amino-3-chloro-1-(b-D-ribofuranosyl)-1H-pyrazolo-
[3,4-d]pyrimidine-6-one (3b)
As described for 3a, 3-chloro-1-(b-D-ribofuranosyl)-1H-pyrazolo-
[3,4-d]pyrimidine-4,6-diamine (1b) (120 mg, 0.38 mmol) was
converted to 3b, yielding a colourless solid (50 mg, 42%) (Found:
C, 37.68; H, 3.65; N, 21.89. C10H12ClN5O5 requires C, 37.81; H,
3.81; N, 22.04%); kmax(MeOH)/nm 293 (e/dm3 mol−1 cm−1 4600),
250 (7300) and 229 (23 800); dH(250 MHz; DMSO-d6; Me4Si) 3.44–
3.56 (2 H, m, 2 × 5ꢀ-H), 3.84 (1 H, m, 4ꢀ-H), 4.08 (1 H, m, 3ꢀ-H),
4.41 (1 H, m, 2ꢀ-H), 4.90–5.34 (3 H, m, 5ꢀ-OH, 3ꢀ-OH and 2ꢀ-OH),
5.81 (1 H, d, J 4.8, 1ꢀ-H), 7.56 (2 H, br s, NH2), 11.23 (1 H, br s,
5-NH).
4-Amino-3-bromo-5-methyl-1-(b-D-ribofuranosyl)-1H-
pyrazolo[3,4-d]pyrimidine-6-one (3ck)
4-Amino-3-bromo-1-(b-D-ribofuranosyl)-1H-pyrazolo-
[3,4-d]pyrimidine-6-one (3c)
To a solution of compound 3c (120 mg, 0.33 mmol) in H2O
(2.5 cm3), trimethylphosphate (2.1 cm3, 18.3 mmol) was added.
The mixture was stirred at 50 ◦C and kept alkaline by addition of
2 N NaOH. After 20 h, the mixture was cooled and diluted with
water (10 cm3) and neutralized. The mixture was then evaporated
and co-evaporated several times with MeOH, and the residue was
adsorbed on silica gel (3 g) and applied to a FC silica gel column
(5 × 2.5 cm, CH2Cl2–CH3OH 50 : 1 → 5 : 1), to give compound 3ce
(20 mg, 16%) from the early fractions, and 3ck (40 mg, 32%) from
the later fractions as a colourless solid (Found: C, 35.30; H, 4.11;
N, 18.55. C11H14BrN5O5 requires C, 35.12; H, 3.75; N, 18.62); TLC
(silica gel, CH2Cl2–CH3OH, 5 : 1): Rf 0.35; kmax(MeOH)/nm 295
(e/dm3 mol−1 cm−1 3500) and 248 (3700); dH(250 MHz; DMSO-d6;
Me4Si) 3.36 (3 H, s, CH3), 3.43–3.66 (2 H, m, 2 × 5ꢀ-H), 3.83 (1 H,
m, 4ꢀ-H), 4.07 (1 H, m, 3ꢀ-H), 4.43 (1 H, m, 2ꢀ-H), 4.90 (1 H, m,
5ꢀ-OH), 5.08 (1 H, d, J 4.8, 3ꢀ-OH), 5.30 (1 H, d, J 6.1, 2ꢀ-OH),
5.76 (1 H, d, J 3.4, 1ꢀ-H), 8.01 (2 H, br s, NH2).
As described for 3a, compound 1c (361 mg, 1 mmol) was
converted to 3c (206 mg, 57%), which was obtained as a
yellowish solid (Found: C, 32.95; H, 3.35; N, 19.15. C10H12BrN5O5
requires C, 33.17; H, 3.34; N, 19.34%); kmax(MeOH)/nm
293 (e/dm3 mol−1 cm−1 4200), 248 (5800) and 231 (22 400);
dH(250 MHz; DMSO-d6; Me4Si) 3.44–3.57 (2 H, m, 2 × 5ꢀ-H),
3.85 (1 H, m, 4ꢀ-H), 4.08 (1 H, m, 3ꢀ-H), 4.45 (1 H, m, 2ꢀ-H), 5.01–
5.60 (3 H, m, 5ꢀ-OH, 3ꢀ-OH and 2ꢀ-OH), 5.80 (1 H, d, J 4.9, 1ꢀ-H),
7.66 (2 H, br s, NH2).
4-Amino-1-(b-D-ribofuranosyl)-3-iodo-1H-pyrazolo-
[3,4-d]pyrimidine-6-one (3d)
As described for 3a, compound 1d (143 mg, 0.35 mmol) was con-
verted to 3d, which was obtained as a white powder (80 mg, 56%)
(Found: C, 29.46; H, 3.05; N, 17.27. C10H12IN5O5 requires C, 29.36;
H, 2.96; N, 17.12%); kmax(MeOH)/nm 295 (e/dm3 mol−1 cm−1
4200) and 235 (19 500); dH(250 MHz; DMSO-d6; Me4Si) 3.40–
3.57 (2 H, m, 2 × 5ꢀ-H), 3.85 (1 H, m, 4ꢀ-H), 4.09 (1 H, m, 3ꢀ-H),
4.45 (1 H, m, 2ꢀ-H), 4.80–5.55 (3 H, m, 5ꢀ-OH, 3ꢀ-OH and 2ꢀ-OH),
5.77 (1 H, s, 1ꢀ-H), 7.51 (2 H, br s, NH2).
Acknowledgements
We thank Dr Yang He, Dr Xiaomei Zhang and Simone Budow
for the NMR spectra, and Dr Peter Leonard and Mr Nhat Quang
Tran for supplying compound 9. We gratefully acknowledge
financial support by Idenix Pharmaceuticals, Cambridge, US, and
ChemBiotech, Mu¨nster, Germany.
4-Amino-3-bromo-6-methoxy-1-(b-D-ribofuranosyl)-1H-pyrazolo-
[3,4-d]pyrimidine (3ce) and 4-amino-3-bromo-7-methyl-1-
(b-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine-6-one (18)
References
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