The synthesis of 7-deazaguanines as potential inhibitors of guanosine triphosphate cyclohydrolase I

Article abstract of DOI:10.1016/j.tet.2003.11.030

Variously substituted 7-deazaguanines are of interest as inhibitors of GTP cyclohydrolase I, the first enzyme in the biosynthetic pathway leading to dihydrofolate and tetrahydrobiopterin. Methods are described for the synthesis of 7-deazaguanines substituted at positions 2, 6 and 9 (purine numbering) such that a wide diversity of compounds can be prepared. These methods supplement our previous work that established routes for the synthesis of 7- and 8-substituted 7-deazaguanines. Emphasis is placed on the properties of 2-thioalkyl pyrimidines as intermediates because they provide the basis for a traceless solid-state synthesis of purines, pteridines, and their analogues. Compounds prepared have been assessed in a primary screen for their ability to inhibit GTPCH I and 8-methyldeazaguanine has been shown to be significantly more potent than any inhibitor yet described. Several compounds appeared to undergo transformation by GTPCH I; with the aid of a model reaction, their behaviour can be interpreted in the context of the mechanism of the hydrolytic phase of GTPCH I.

Full text of DOI:10.1016/j.tet.2003.11.030

Tetrahedron 60 (2004) 943–959
The synthesis of 7-deazaguanines as potential inhibitors of
guanosine triphosphate cyclohydrolase I
Colin L. Gibson,a Salvatore La Rosa,a Kyuji Ohta,b Peter H. Boyle,c Fabien Leurquin,c
Alexandra Lemac¸onc and Colin J. Sucklinga
,
*
aDepartment of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, Scotland, UK
bDepartment of Chemistry, Hosei University, Tokyo, Japan
cUniversity Chemical Laboratory, Trinity College, Dublin, Ireland
Received 4 September 2003; revised 20 October 2003; accepted 13 November 2003
Abstract—Variously substituted 7-deazaguanines are of interest as inhibitors of GTP cyclohydrolase I, the first enzyme in the biosynthetic
pathway leading to dihydrofolate and tetrahydrobiopterin. Methods are described for the synthesis of 7-deazaguanines substituted at
positions 2, 6 and 9 (purine numbering) such that a wide diversity of compounds can be prepared. These methods supplement our previous
work that established routes for the synthesis of 7- and 8-substituted 7-deazaguanines. Emphasis is placed on the properties of 2-thioalkyl
pyrimidines as intermediates because they provide the basis for a traceless solid-state synthesis of purines, pteridines, and their analogues.
Compounds prepared have been assessed in a primary screen for their ability to inhibit GTPCH I and 8-methyldeazaguanine has been shown
to be significantly more potent than any inhibitor yet described. Several compounds appeared to undergo transformation by GTPCH I; with
the aid of a model reaction, their behaviour can be interpreted in the context of the mechanism of the hydrolytic phase of GTPCH I.
q 2003 Elsevier Ltd. All rights reserved.
Guanosine triphosphate cyclohydrolase I (GTPCH I) is the
first enzyme in the biosynthetic pathway leading to
dihydrofolate and the pathway leading to tetrahydrobiop-
terin, two essential metabolic cofactors.1 Recent clarifica-
tion of the crystal structure of GTPCH I2 has demonstrated
the presence of a zinc cation at the active site and assigned it
a role in acid–base catalysis of the hydrolytic opening of the
purine ring. This information has strengthened the infor-
mation for the design of inhibitors of GTPCH I. Such
inhibitors may have value in antibacterial therapy or in
agrochemistry.3 It is notable that several antibiotics
containing deazaguanines as aglycones have been discov-
ered.4 In a previous paper in this series5 we described
versatile syntheses of 7-deazaguanines with substituent
diversity at positions 7 and 8. Here we describe extensions
of these methods to provide diversity at positions 2, 6 and 9
(purine numbering). Taken together, the two papers provide
a large part of the basis for a versatile solid phase synthesis
methodology for purines, pteridines, and their analogues.6
Without ability to tolerate a wide range of substituents,
these methods would be severely limited. We have therefore
studied the capacity of these synthetic methods to prepare a
range of highly substituted deazaguanines. The compounds
prepared have been assayed against GTP-cyclohydrolase 1.
New inhibitors have been identified and unexpected
reactivity of several compounds sheds light on the
mechanism of action of the enzyme.
1. Synthesis of 9-substituted 7-deazaguanines
In our previous work,5 N(9)-alkyl groups, alternatives to
ribose, were introduced by substitution of 2-amino-6-
chloro-4(3H)-pyrimidinone 1 (Scheme 1a). It is important
for obtaining significant biological activity to be able to
include polyfunctional substituents, especially hydroxyl
groups that might be phosphorylated in vivo. Consequently,
the substitution strategy was extended in this work to
include 3-aminopropane-1,2-diol and 4-aminobutan-1-ol as
substituents; the corresponding pyrimidines 2b and 2c
underwent smooth cyclisation with chlorocyanoacetalde-
hyde to afford the 7-cyano-7-deazaguanines 3b and 3c in
60–70% yield. However, this route was shown to be limited
to primary amines lacking a-substituents; none of ribosyl-
amine, cyclopentylamine, cyclohexylamine or 1,2-dihy-
droxy-3-amino-5-hydroxymethylcyclopentane reacted with
the 6-chloropyrimidine. We also showed in our previous
work5 that a range of 8-substituted 9-alkyl deazaguanines 4
could be prepared from alkyldiaminopyrimidines such as 2
and the oximes of a-haloketones in the presence of base via
intermediate 5-substituted oximes.
Keywords: Purine analogues; Synthesis; Deazaguanines; GTP
cyclohydrolase 1; Inhibition; Mechanism.
*
Corresponding author. Tel.: þ44-0141-548-2271; fax: þ44-0141-548-
4822; e-mail address: c.j.suckling@strath.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.11.030

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C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
Scheme 1. R¼a H, b CH2CH(OH)CH2OH, c (CH2)4OH, d CH2CHvCH2. Reagents: (i) CHCl(CN)CHO; (ii) R1C(vNOH)CH2Br, Et3N; (iii) Hþ, PhCHO;
(iv0 CH2vCHCH2Br, K2CO3; (v) 6,6-dimethyl-5,7-dioxaspiro[2.5]octane-4,8-dione, K2CO3; (vi) NaBH4, t-BuOH; (vii) NBS.
In order to insert substituents more similar to ribose, it was
necessary, therefore, to investigate direct alkylation of N-9
as an alternative strategy (Scheme 1b). Whilst this strategy
is effective with guanosines,7 we have found that with
deazaguanines there are difficulties associated with the
several possible sites of alkylation on nitrogen and oxygen.4
It was therefore prudent to establish that selective alkylation
at N9 was indeed possible. This was done by alkylating the
7-cyano-7-deazaguanine 3a with allyl bromide in DMF in
the presence of potassium carbonate, which afforded a
single compound, 3d, in 68% yield after purification. That
alkylation had taken place exclusively at N9 was confirmed
by synthesis of 3d from chloropyrimidine 1 by reaction of
the latter with allylamine and subsequent cyclisation. The
material thus obtained was identical in all respects to that
prepared by direct alkylation. Alkylation with highly
functionalised reagents to provide closer analogues to
ribose was therefore attempted, based upon published
syntheses of the drugs penciclovir and famciclovir.8 For
clean alkylation at N9, a reactive electrophile was required.
Triethyl 3-bromopropane-1,1,1-tricarboxylate failed to
react on extended reaction times but 6,6-dimethyl-5,7-
dioxaspiro[2.5]octane-4,8-dione alkylated 7-cyano-7-
deazaguanine 3a to afford the derivative 5 in 65% yield.
Reduction of the diester was accomplished with sodium
borohydride affording the corresponding deazaguanine diol,
6, substituted by an acyclic ribose analogue, in 86% yield.
This compound was brominated at C8 with N-bromo-
succinimide to give 7 and access to the range of sulfur-
containing derivatives described by us previously.5 It is
probable that the 7-cyano group is important in controlling
the selectivity in these reactions by delocalisation of an
anion formed at N9. Similar reactions attempted with
8-ethoxycarbonyl-7-deazaguanine (4, R¼H, R1¼CO2Et)
led to complex mixtures; in that case, it seems that
introduction of the alkyl group at the pyrimidine stage is
essential.
2. Substitution at C2
It is well known that the amino–oxo substitution pattern of
the pyrimidine ring of pteridines and guanosines leads not
only to ambident reactivity but also to experimental

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
945
Scheme 2. R¼a CH2Ph, b Me. Reagents: (i) CF3COCH2Br, EtOH, 60 8C, 17 h; (ii) BrCH2C(vNOH)CO2Et, Et3N, DMF, room temperature, 5 h; (iii) PhCHO,
Hþ, EtOH, reflux, ,12 h.
problems of low solubility. With a view to extension to solid
phase methodologies, the cyclisation chemistry of pyrimi-
dine thioethers was investigated (Scheme 2). 2-Benzylsul-
fanyl- and 2-methylsulfanyl-6-amino-4(3H)-pyrimidinones
(9a,b) were prepared by direct alkylation of the correspond-
ing thiol 8. Surprisingly, compounds 9a and 9b failed to
give purines or pyridopyrimidines when treated with
C-electrophiles such as chloroacetaldehyde, bromo-
acetaldehyde diethyl acetal, chlorocyanoacetaldehyde,
bromomalonate, ethyl acrylate, acetylacetone, diethyl
oxalate, ethyl glyoxylate, and ethyl 2,4-dioxopentanoate,
in DMF or ethanol in the presence of base (triethylamine) or
acid (4-toluenesulfonic acid) under reflux for periods of up
to 4 days.
10 in 40% yield, a reaction course observed previously with
this reagent.9 Ethyl bromopyruvate oxime, on the other
hand, did give the desired C-5 alkylated pyrimidine 11 when
reacted with 9a, and the product 11 was cyclised under our
normal conditions by transoximation with benzaldehyde
and acid catalysis to afford the 8-ethoxycarbonyl deaza-
guanine 12 in 81% yield. The influence of the C2 substituent
in the reactivity of pyrimidines in this series is striking.
Although carbon electrophiles show very limited reactivity,
we have shown5,10 that nitrosation occurs readily leading to
effective syntheses of sulfanyl pteridines and purines.
3. Substitution at C6 (C4 in pyrrolo[2,3-d] numbering)
Only two reagents were found to react. Bromotrifluoro-
acetone reacted with 9a to afford the furo[2,3-d]pyrimidine
Derivatisation of the 4-oxo function in pyrimidines has been
found to be a versatile method of controlling reactivity. This
Scheme 3. Reagents: (i) POCl3, pyridine, reflux, overnight; (ii) TFAA, pyridine, ClC6H4SH, NH4OH, H2O2, 0 8C; (iii) Ac2O, DMAP, reflux, 6 h.

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C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
Scheme 4. Reagents: (a) (i) PhCH2SH, NaOH, aq. EtOH, 80 8C, overnight; (ii) PhCH2Cl; (iii) CH3C6H4SH, NaOH, aq. EtOH; (b) (iv) ClCH2CHO, 1 equiv.
50 8C, overnight; (v) ClCH2CHO, 2 equiv., 50 8C, 22 h; (vi) Cl(CN)CHCHO, 50 8C, overnight; (vii) bromopyruvate oxime, Et3N, 80 8C, 10 h, then Hþ,
PhCHO; (c) (viii) PhCH2SH, NaOH, room temperature, 10 min, (ix) CH2vCHCH2Br, K2CO3, 100 8C, 5 days; (x) Bredereck’s reagent, 60 8C, 10 min.

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
947
modification was applied both to 7-deazaguanines them-
selves and to the precursor pyrimidines but led to a variety
of undesired cyclisations (Schemes 3 and 4) as described
below. Conversion of 7-cyano-7-deazaguanine 3a into more
soluble, lipophilic derivatives was approached by chlorin-
ation with phosphorus oxychloride to afford 13 in modest
yield. Preferably, therefore, 13 was treated sequentially with
trifluoroacetic anhydride and pyridine, 4-chlorothiophenol,
ammonium hydroxide, and hydrogen peroxide to give the
(4-chlorophenylsulfanyl)-deazaguanine 14 in 52% overall
yield.8 Other reagents investigated included acetic
anhydride/DMAP; with one equivalent, monosubstitution
at N9 giving 15 was observed but used in excess, 3,9-
diacetyl-7-deazaguanine 16 was obtained.
nucleophilic reactivity of the 4-amino group, an attempt was
made to modify the reactivity to obviate these problems
(Scheme 4c). Accordingly 2-amino-4,6-dichloropyrimidine
was converted into the benzylsulfanyl ether 25 (96%) and a
sterically hindered amino group introduced by substitution
of the second chloride with allylamine to give 26 (45%).
The remaining amino group was protected with a strongly
electron donating dimethylaminoformimino group using
Bredereck’s reagent to give the required modified pyrimi-
dine 27 (87%). Disappointingly, C5 substitution was not
observed with chloracetaldehyde; cyclisation on N3 and the
allylamine occurred to give 28, analogous to the previous
reactions but without dehydration. It must therefore be
concluded that the synthesis of deazaguanines using 2- and
4-pyrimidine sulfanyl ethers is severely limited to
2-sulfanyl systems with electrophiles derived from a-halo-
oximes.
In view of the problems associated with C6 substitution
described above, it was important to extend the investi-
gation of sulfanyl ethers to C4 in pyrimidines (Scheme 4a).
2,4-Diamino-6-chloropyrimidine readily underwent substi-
tution with benzyl mercaptan to afford the benzyl sulfanyl
ether 20a in 85% yield and the same compound was
obtained by alkylation of 2,4-diaminopyrimidine-6-thiol
with benzyl chloride. With extension to solid phase
synthesis in mind and the evidence that thiophenyl
ethers are easier to cleave than thiobenzyl ethers,11 the
(4-methylphenyl)sulfanyl ether 20b was also prepared.
Disappointingly, the 4-sulfanyl ethers introduced yet
another reactivity pattern into the pyrimidine ring. Once
again, substitution at C5 was not observed. Instead,
cyclisation between N3 and 4-NH2 occurred (Scheme 4b).
This was unambiguously demonstrated using chloro-
acetaldehyde (1 equiv.) which afforded the imidazo[1,2-c]-
pyrimidine 21 from 20a in low yield (25%), the structure
being confirmed by NMR and X-ray crystallography.
Excess chloroacetaldehyde led to dicyclisation and 22.
Chlorocyanoacetaldehyde gave the cyanoimidazo[1,2-c]-
pyrimidine 23. Even the highly reactive bromopyruvate
oxime in the presence of triethylamine, which had given
C-substitution in the 2-sulfanyl series, reacted on nitrogen to
form 24.
4. Ribosyl derivatives of 7-deazaguanines
A second potential benefit of sulfanyl ethers is the
simplification of alkylation chemistry. The synthesis of
deazaguanine glycosides in particular might be facilitated
by the reduced nucleophilicity of the sulfanyl derivatives
compared with their amino and oxo analogues. a-1-
Bromoribose-2,3,5-tribenzoate12 was reacted with the
4-chlorophenyl sulfanyl ether 14 under phase transfer
conditions but no riboside formation was observed.
However riboside formation was successful using 7-deaza-
guanine 3d and the 7-cyano analogue 3a using 1-acetoxy-
ribose-2,3,5-tribenzoate with Lewis acid catalysis13
affording 29 or 30, respectively as a mixture of anomers
in each case (Scheme 5). It was not found possible to
separate the anomers and, since interesting biological
activity and structural possibilities were found with simpler
compounds, no development work was carried out on
glycosylation reactions.
5. Leads from molecular modelling
Arguing that this greatly diminished reactivity at C5 was
due to a pronounced decrease in electron density and to high
As part of our international collaboration, Professor Kyuji
Scheme 5. Ribosyl derivatives of deazaguanines. Reagents: hexamethyldisilazane, TMSCl, SnCl4, MeCN, 21 h room temperature.

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C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
Ohta in Japan has been investigating the potential binding of
deazaguanines and other compounds to the active site of
GTPCH I. His studies included the zinc ion and led to the
suggestion that deazaguanines with carboxyl or thiol
substituents might be very tight binding inhibitors thereby
obviating the need for phosphorylated derivatives.14
Accordingly, target molecules represented by the deaza-
guanine carboxylates with simplified ribose analogues 31,
and 32 were identified and their synthesis developed
(Scheme 6). The precursor pyrimidine 33 was prepared by
the conventional substitution methods and cyclised with
each of the two established reagents, chlorocyanoacetalde-
hyde and ethyl bromomalonate oxime. In the former case, a
mixture of acetal 34a and deprotected deazaguanine 34b in
a ratio of 2.5:1 was obtained. Hydrolysis of the cyano group
with potassium hydroxide afforded the mixture of car-
boxylic acids 35a and 35b and esterification with thionyl
chloride and methanol gave one of the target compounds 31.
In the latter case, C5 substitution occurred without loss of
the protecting group to give 36 but we were unable to find
conditions under which cyclisation to the deazaguanine 32
could be achieved without cleavage of the protecting group.
However, the ester 32 was obtained in 62% yield on
cyclisation of 36 thereby confirming the practical utility of
the nitroso alkene route for the synthesis of polyfunctional
8,9-substituted deazaguanines.17
compounds were screened. The compound set included a
wide variety of substituted pyrimidines, purines, pteridines,
and 7-deazapurines that were synthesised in the present
work and in previous studies.17 The UV assay was carried
out by adding a solution of the test compound dissolved in
DMSO to a solution of GTPCH I in KCl/Tris buffer, pH 8.5,
initiating the reaction by the addition of enzyme. Reaction
progress was monitored over 3 min at 1 s intervals by
following the absorption at 330 nm. At this wavelength, the
product 7,8-dihydroneopterin triphosphate absorbs but the
substrate GTP does not. Control experiments were carried
out under the same conditions in the absence of test
compound and to demonstrate that DMSO had no effect on
the reaction in the concentrations used. The HPLC assay
monitored the formation of 7,8-dihydroneopterin tri-
phosphate by separation of it on the HPLC column from
other reaction components and integration of the product
peak. For reasons of availability and activity of GTPCH I, it
was not possible to undertake full kinetic assays of the
compounds prepared. The available assays, however,
functioned together as an effective screen to identify
compounds that were significant inhibitors and compounds
that underwent reaction with GTPCH I.
The assays showed that 7-deazapurines, substituted at
position 8 but unsubstituted at positions 7 and 9, had
behaviour consistent with some form of inhibition on
GTPCH I. In particular, the HPLC assay showed that in
the presence of 8-methyl-7-deazaguanine 37b (2.9 mM), the
amount of 7,8-dihydroneopterin formed from GTP was
only 6% of the amount formed in the absence of 37b,
under otherwise identical conditions. The most widely
discussed inhibitor, DAP, by comparison showed only 58%
inhibition under these conditions. Other related
compounds showed similar but smaller decreases: 8-tri-
fluoromethyl-7-deazaguanine 37c 17%, 8-azaguanine 43%,
and 8-ethoxycarbonyl-7-deazaguanine 78%. This general
behaviour has been confirmed independently and using a
different method (Dr Christian Hesslinger, Frankfurt18).
8-Methyl-7-deazaguanine 37b thus appears to be the
most potent inhibitor of GTPCH 1 to have been described
so far.
6. 7-Deazaguanines as inhibitors of GTPCH I
We have undertaken assays of the activity of compounds
prepared in our studies as inhibitors of GTPCH I using
screens designed for rapid assay using small quantities of
scarce enzyme. Known inhibitors include guanine,
8-hydroxy-, 8-methyl-, 8-mercapto-, and 8-bromo-
guanine,15 8-azaguanine, and what is probably the most
widely used inhibitor, 2,6-diamino-4(3H)-pyrimidinone
(DAP).16 GTPCH I is also inhibited naturally by tetra-
hydrobiopterin mediated by GTPCH I feedback regulatory
protein.15 We have developed two screening assays, one
based on UV and the other on HPLC, both of them being
suitable for a high throughput of samples and a total of 52

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
949
Scheme 6. Reagents: (i) RNH2, Et3N, EtOH, reflux, 2 days; (ii) Cl(CN)CHCHO, aq. NaOAc, 50 8C, 20 h; (iii) aq. KOH, reflux, 5 h; (iv) AcOH; (v) SOCl2,
MeOH, 50 8C, 3 h; (vi) Et3N, DMF, room temperature, 6 h; (vii) HCl, aq. EtOH, PhCHO, reflux, 2 days.
Observations in the UV assay suggested that 37b and related
compounds may react with the GTPCH I to form a new
product, other than 7,8-dihydroneopterin triphosphate. Even
in the absence of GTP, the absorption at 330 nm increased
over the first minute of assay before slowly decreasing
during the remainder of the observation period. Compounds
37a, 37b, 37c, 38a, and 38b all showed this effect in the UV
assay. A possible explanation of the apparent reaction of
these compound with GTPCH I is that, through its normal
mechanism, GTPCH I is causing hydrolytic opening of the
pyrazole ring. In the case of 38b this would lead to
compound 39. To determine whether this proposed pathway
had a reasonable basis in laboratory chemistry, the ester 38b
was incubated in d6-DMSO in the presence of sodium
ethoxide and the NMR spectrum monitored over many days;
special precautions to dry the solvent and reagents were not
taken. The use of sodium ethoxide under these conditions
provided for the presence of a low concentration of
hydroxide thereby avoiding more extensive hydrolytic
decomposition. A slow reaction was clearly observed,
most notably from the downfield region of the spectrum,
where a new peak at d 8.51 was consistent with the presence
of a formamide and a peak at d 7.05 indicative of the
presence of a deshielded alkene (Fig. 1). After 100 days,
integration suggested that about 20% conversion into this
compound 39 had occurred. A reasonable interpretation of
these results (Scheme 7) would be in terms of formation of
the ring opened formamide 39. These preliminary
observations encourage further work on the properties and
reactivity of deazaguanines with GTPCH I.
7. Conclusions
In this work and in our previous study5 we have defined the
limitations inherent in the ring synthesis of deazaguanines

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C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
Scheme 7. Hypothesis for reaction of deazaguanines with GTPCH I.
from substituted pyrimidines and have shown that within
these limitations, a wide range of structural diversity can be
accessed. We have developed chemistry potentially
applicable to solid phase synthesis of a range of related
bicyclic heterocyclic compounds.5 Importantly, we have
adduced preliminary evidence that deazaguanines do indeed
inhibit and react with GTPCH I in ways that are of interest
in the context of the mechanism of action of the enzyme.
Moreover our most recent results indicate that deaza-
guanines are effective in modulating the activity of pteridine
biosynthesis in cellular as well as purified enzyme
systems.18
Figure 1. 400 MHz spectrum of 40b after reacting for 100 days with NaOEt in DMSO solution.

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
951
8. Experimental
pyrimidin-4(3H)-one 1 (1.13 g, 6.93 mmol) in water
(3 mL), was added ethanol (20 mL), triethylamine (2 mL)
and 1-amino-4-butanol (1.6 g, 18.4 mmol). The mixture was
heated to reflux for 2 days, then the solvent was evaporated
under reduced pressure to give a colourless solid that was
washed with water, ethanol and diethyl ether, dried under
reduced pressure, to afford the title compound 2c (0.95 g,
4.9 mmol, 70%; 206–208 8C (lit.21 206 8C)). Found: HRMS
(FAB) 199.1194; C8H15N4O2 (Mþ1) requires 199.1195. dH
(DMSO) 1.38–1.47 (4H, m, C(10)H2, C(11)H2), 3.01 (2H,
br s, C(12)H2), 3.34–3.40 (2H, m, C(9)H2), 4.40 (1H, s,
C(5)H), 4.41 (1H, br s, OH), 6.12 (2H, br s, NH2), 6.33 (1H,
br s, N(8)H), 9.66 (1H, br s, N(3)H).
8.1. Instrumentation and general materials
NMR spectra were determined on a Bruker Spectrospin
spectrometer operating at 400 MHz for 1H spectra and
100 MHz for 13C spectra. Chemical shifts are reported as
ppm relative to TMS measured from the solvent resonance.
IR spectra were determined using a Mattson 1000 FT
spectrometer or a Nicolet Impact 400D FT spectrometer.
Mass spectra were measured on a Jeol JMS AX505
spectrometer. Microanalyses were carried out using a
Perkin–Elmer Series II instrument at the University of
Strathclyde. UV spectra were determined using a Perkin–
Elmer Lambda 2 spectrometer. Melting points, when
measurable, were determined on a Reichert hot stage
apparatus and are uncorrected. TLC was carried out on
silica (Merck 0.25 mm 60 F254). Column chromatography
was carried out using silica gel (230–400 mesh;
40–60 mm).
8.1.4. 2-Amino-7-(4-hydroxybutyl)-4-oxo-4,7-dihydro-
3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 3c. To
a
suspension of 2-amino-6-[(4-hydroxybutyl)amino]-4(3H)-
pyrimidinone 2c (1.10 g, 5.5 mmol) and sodium acetate
(1.03 g) in water (20 mL) was added chloro(formyl)aceto-
nitrile16 (2 g, 19.4 mmol). Upon addition, the solution
became green, turning deep blue immediately. The reaction
was stirred at 50 8C for 2 days. The solution was filtered and
the colourless solid obtained was washed with water,
ethanol, and diethyl ether and dried under reduced pressure
to afford the title compound 3c (1.14 g, 4.7 mmol, 84%; mp
.260 8C). Found: HRMS (FAB) 248.1138, C11H14N5O2
(Mþ1) requires 248.1147. dH (DMSO) 1.30–1.37 (2H, m,
C(11)H2), 1.68–1.76 (2H, m, C(12)H2), 3.31–3.39 (2H, m,
C(13)H2), 3.92–3.96 (2H, m, C(10)H2), 4.40–4.43 (1H, m,
OH), 6.49 (2H, br s, NH2), 10.70 (1H, s, N(3)H) dC (DMSO)
26.66 (C-11), 29.81 (C-12), 44.85 (C-13), 60.56 (C-10),
85.06 (C-5), 99.21 (C-4a), 116.03 (CN), 130.68 (C-6),
151.07, 154.12, 157.80 (C-4, C-7a, C-2). nmax (KBr) 3343
(NH2), 3182, 2228 (CN), 1681, 1645, 1599, 1548, 1423,
Reaagents were bought from Aldrich (Gillingham, Dorset,
UK).
8.1.1. 2-Amino-6-[(2,3-dihydroxypropyl)amino]-4(3H)-
pyrimidinone 2b. To a suspension of 2-amino-6-chloro-
pyrimidin-4(3H)-one 1 (1.130 g, 6.93 mmol) in water
(2.8 mL), was added ethylene glycol dimethyl ether
(10 mL) and 1-amino-2,3-propandiol (1.7 g, 18.7 mmol).
The mixture was heated to reflux for 6 h and then the solvent
was evaporated under reduced pressure to give the required
pyrimidinone 2b as a colourless solid (0.927 g, 4.63 mmol,
68%). After washing successively with water, ethanol and
diethyl ether and drying under reduced pressure, the product
had mp 204–206 8C. Found: HRMS (FAB) 201.0980;
C7H13N4O3 (Mþ1) requires 201.0988. dH (DMSO) 2.96
(1H, m, C(9)H), 3.16 (2H, m, C(10)H2), 3.52 (2H, m,
C(8)H2), 4.47 (1H, s, C(5)H), 4.55 (1H, s, C(10)OH), 4.77
(1H, s, C(9)OH), 6.14 (3H, br s, N(7)H2, N(6)H), 9.66 (1H,
br s, N(3)H). dC (DMSO) 44.77 (C-8), 64.23 (C-10), 70.65
(C-9), 75.71 (C-5), 155.33 (C-2), 163.42 (C-4), 164.77
(C-6). nmax (KBr) 3425, 1670, 1537, 1455, 1256, 984,
1343, 1101, 785 cm21
.
8.1.5. 6-(Allylamino)-2-amino-4(3H)-pyrimidinone 2d.
To a suspension of 2-amino-6-chloropyrimidin-4(3H)-one
56 (1.13 g, 6.93 mmol) in water (3 mL), was added ethylene
glycol dimethyl ether (10 mL) and allyl amine (1.04 g,
18.4 mmol). The mixture was heated to reflux for 6 h, then
the solvent was evaporated under reduced pressure to give a
colourless solid that was washed with water, ethanol and
diethyl ether and dried under reduced pressure to afford the
title compound 2d (0.68 g, 4.1 mmol, 60%; mp 196–
198 8C). Found: HRMS (EI) 166.0860: C7H10N4O requires
166.0855. dH (CDCl3) 3.68 (2H, s, C(9)H2), 4.41 (1H, s,
C(5)H), 5.04 (1H, d, J¼9.2 Hz, 1£C(11)H2), 5.13 (1H, d,
J¼17.2 Hz, 1£C(11)H2), 5.75–5.84 (1H, m, C(10)H), 6.15
(2H, br s, N(7)H2), 6.52 (1H, br s, N(8)H), 9.75 (1H, br s,
N(3)H). dC (DMSO) 45.45 (C-9), 77.89 (C-5), 117.66
(C-11), 134.11 (C-10), 156.23 (C-2), 162.59 (C-4), 165.07
(C-6).
783 cm21
.
8.1.2. 2-Amino-7-(2,3-dihydroxypropyl)-4-oxo-4,7-di-
hydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 3b.
To a suspension of 2-amino-6-[(2,3-dihydroxypropyl)-
amino]-4(3H)-pyrimidinone 3b (0.912 g, 4.56 mmol) and
sodium acetate (0.94 g, 10 mmol) in water (30 mL), was
added freshly prepared chloro(formyl)-acetonitrile19
(0.54 g, 5.24 mmol). The mixture was left stirring for 20 h
at 50 8C. A precipitate was collected by filtration, washed
with water, methanol and diethyl ether to give the required
pyrrolopyridine 3b as a dark solid (0.32 g, 1.3 mmol, 30%;
mp .260 8C (lit.20 276–278 8C)). Found: HRMS (EI) found
215.0807 (M22£OH); C10H11N5O3 requires 249.0862. dH
(DMSO) 3.26 (2H, m, C(12)H2), 3.75 (2H, m, C(10)H2),
4.10 (1H, m, C(11)H), 4.74 (1H, s, C(12)H2OH), 4.99 (1H,
s, C(11)HOH), 6.50 (2H, s, N(8)H2), 7.59 (1H, s, C(6)H),
10.73 (1H, s, N(3)H).
8.1.6. 7-Allyl-2-amino-4-oxo-4,7-dihydro-3H-pyr-
rolo[2,3-d]pyrimidine-5-carbonitrile 3d. (a) Using allyl
bromide. To 7-cyano-7-deazaguanine 3a (0.5 g, 2.8 mmol)
in anhydrous DMF (40 mL) was added allyl bromide
(0.35 g, 2.9 mmol) and potassium carbonate (0.45 g,
3.2 mmol). The reaction was left stirring for 4 days at
100 8C, then filtered and evaporated under reduced pressure.
The residue was dissolved in water and extracted with
dichloromethane, dried over anhydrous sodium sulphate,
8.1.3. 2-Amino-6-[(4-hydroxybutyl)amino]-4(3H)-pyri-
midinone 2c. To a suspension of 2-amino-6-chloro-

952
C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
and evaporated to give the product 3d as pale a yellow solid
(0.15 g, 0.8 mmol, 27%; mp .260 8C). Found: HRMS (EI)
215.0792, C10H9N5O requires 215.0807. dH (DMSO) 4.58
(2H, d, J¼4.8 Hz, C(10)H2), 4.95 (1H, d, J¼17.2 Hz,
1£C(12)H2), 5.17 (1H, d, J¼10.4 Hz, 1£C(12)H2),
5.92–6.01 (1H, m, C(11)H), 6.52 (2H, br s, N(8)H2),
10.76 (1H, s, N(3)H). dC (DMSO) 46.85 (C-10), 85.52
(C-5), 99.13 (C-4a), 115.87 (C-9), 117.63 (C-12), 130.49
(C-6), 133.73 (C-11), 151.07, 154.27, 157.82 (C-4, C-7a,
C-2). nmax (KBr) 3416, 3325, 2224, 1673, 1626, 1176,
which was purified by column chromatography on silica gel
eluting with ethyl acetate/methanol 100/0 up to 0/100. The
product 6 (0.556 g, 2.0 mmol, 86%) was collected from the
last fractions as a pale yellow solid, mp .260 8C. Found:
HRMS (FAB) 278.1247, C12H16N5O3 (Mþ1) requires
278.1253. dH (DMSO) 1.39–1.44 (1H, m, C(12)H), 1.66
(2H, q, J¼7.2 Hz, C(11)H2), 3.30–3.43 (4H, m, C(14)H2,
C(13)H2), 4.00 (2H, t, J¼7.2 Hz, C(10)H2), 4.50 (2H, s,
2£OH), 6.86 (2H, s, N(8)H2), 7.71 (1H, s, C(6)H), 11.04
(1H, br s, N(3)H). dC (DMSO) 29.05 (C-11), 41.10 (C-12),
43.29 (C-10), 61.57 (C-13, C-14), 85.05 (C-5), 97.14
(C-4a), 116.12 (C-9), 130.49 (C-6), 151.62, 154.41,
157.60 (C-2, C-7a, C-4). nmax (KBr) 3417 (NH2), 3350,
3233 (NH), 2940 (CH), 2227 (CN), 1683 (CvO), 1637,
929 cm21
.
(b) Using allyl amine. To a suspension of 6-(allylamino)-2-
amino-4(3H)-pyrimidinone 2d (0.68 g, 4 mmol) and sodium
acetate (0.9 g) in water (20 mL) was added chloro(formyl)-
acetonitrile (1.2 g, 11.6 mmol). This was left stirring at
50 8C overnight. After the solution was filtered and the solid
obtained was washed with water, ethanol, and diethyl ether
to afford the product that was found to be identical with the
product prepared by the method (a) above (0.4 g,
1.86 mmol, 45%).
1597, 1428, 1031, 778 cm21
.
8.1.9. 2-Amino-6-bromo-7-[4-hydroxy-3-(hydroxy-
methyl)butyl]-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyri-
midine-5-carbonitrile 7. To a suspension of 2-amino-7-[4-
hydroxy-3-(hydroxymethyl)butyl]-4-oxo-4,7-dihydro-3H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile 6 (0.556 g,
2.07 mmol) in dry DMF (20 mL), was added N-bromo-
succinimide (0.398 g, 2.24 mmol). The mixture was stirred
at room temperature for 6 h, then evaporated to dryness
under reduced pressure and the residue was triturated with
water (10 mL). The resulting solid was filtered off, washed
with water, ethanol, and then diethyl ether and dried under
reduced pressure to afford the product 7 as a microcrystal-
line off-white solid (0.22 g, 0.61 mmol, 30%; mp 195–
199 8C). Found: HRMS (EI) 357.0258, C12H14N5O831Br
requires 357.0259. dH (DMSO) 1.46–1.51 (1H, m, C(12)H),
1.64 (2H, q, J¼7.6 Hz, C(11)H2), 3.35–3.42 (4H, m,
C(13)H2, C(14)H2), 4.08 (2H, t, J¼7.6 Hz, C(10)H2), 4.41
(2H, s, 2£OH), 6.61 (2H, br s, N(8)H2), 10.88 (1H, s,
N(3)H). dC (DMSO) 28.60 (C-11), 41.10 (C-12), 41.92 (C-
10), 61.74 (C-13, C-14), 79.71 (C-5), 107.38 (C-9), 113.95
(C-4a), 125.83 (C-6), 154.46 (C-2), 156.88 (C-7a). nmax
(nujol) 3342 (NH2), 3222, 2942 (CH), 2229 (CN), 1680,
8.1.7. Dimethyl 2-[2-(2-amino-5-cyano-4-oxo-3,4-di-
hydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl] malonate
5. A mixture of 7-cyano-7-deazaguanine 3a (0.5 g,
2.8 mmol),
2,2-dimethyl-1,3-dioxaspiro[5.2]octane-4,6-
dione (0.65 g, 3.8 mmol), and potassium carbonate
(0.75 g) in dry DMF (20 mL) was stirred at 55 8C under
an atmosphere of N2 for 4 days. The resulting mixture was
filtered and evaporated under reduced pressure to give a
brown solid (the potassium salt). This was dissolved in
methanol (35 mL) saturated with HCl and a precipitate
formed. A further portion of methanol (35 mL) was added
and the mixture was left stirring at room temperature
overnight. The solution was then filtered, evaporated under
reduced pressure and the residue was dissolved in acetone.
Silica gel column chromatography eluting with ethyl
acetate/methanol 5/1, gave the product 5 as a colourless
solid (0.3 g, 0.83 mmol, 30%; mp .260 8C). Found: HRMS
(FAB) 334.1147, C14H16N5O5 (Mþ1) requires 334.1151.
dH (DMSO) 2.26 (2H, q, J¼6.9 Hz, C(11)H2), 3.43 (1H, t,
J¼6.9 Hz, C(12)H), 3.62 (6H, s, 2£C(16)H3), 4.01 (2H, t,
J¼6.9 Hz, C(10)H2), 6.47 (2H, br s, N(8)H2), 7.65 (1H, s,
C(6)H), 10.73 (1H, s, N(3)H). dC (DMSO) 28.81 (C-11),
42.72 (C-10), 48.70 (C-12), 52.92, 52.75 (C-16, C-14),
85.45 (C-5), 99.21 (C-4a), 115.87 (C-9), 130.51 (C-6),
151.22, 154.14, 157.74 (C-4, C-7a, C-2), 169.13 (C-15,
C-13). IR nmax (KBr) 3432 (NH2), 2232 (CN), 1720 (CvO),
1640, 1562, 1400, 1039, 780 cm21
.
8.1.10. 6-Amino-2-(benzylsulfanyl)-4(3H)-pyrimidinone
9a. 6-Amino-2-sulfanyl-4(3H)-pyrimidinone monohydrate
8 (8 g, 50 mmol) was suspended in a mixture of water
(30 mL) and ethanol (50 mL). Triethylamine (10 g, 0.1 mol)
was added and the solution became clear. Benzyl chloride
(7 g, 55 mmol) was added to the stirring solution. Within a
few minutes, an exothermic reaction started with formation
of a colourless precipitate. Stirring was continued for
30 min, the mixture was cooled to 4 8C and the precipitate
was filtered and washed with water and diethyl ether to
afford the title compound 8a (10.5 g, 45 mmol, 90%; 248–
253 8C (lit.22 250–252 8C)). Found: HRMS (EI) 233.0614,
C11H11N3OS requires 233.0623. dH (DMSO) 4.33 (2H, s,
C(7)H2), 4.96 (1H, s, C(5)H), 6.54 (2H, br s, N(12)H2),
7.21–7.32 (3H, m, 2£C(10)H, C(11)H), 7.41–7.43 (2H, m,
2£C(9)H), 11.48 (1H, br s, N(3)H).
1685, 1630, 1427, 787 cm21
.
8.1.8. 2-Amino-7-[4-hydroxy-3-(hydroxymethyl)butyl]-
4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-car-
bonitrile 6. Dimethyl 2-[2-(2-amino-5-cyano-4-oxo-3,4-
dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]malonate 5
(0.83 g, 2.4 mmol) was partially dissolved in t-butanol
(30 mL) at 60 8C under an atmosphere of dry nitrogen.
Sodium borohydride (0.456 g, 12 mmol) was added and the
mixture was heated under reflux while methanol (4 mL) was
added dropwise over 1.75 h. The mixture was then cooled,
methanol (40 mL) was added, and after the effervescence
had ceased, the solvents were evaporated. The residue was
dissolved in water (30 mL) and the solution was neutralised
with 1 M HCl. Evaporation afforded a colourless solid,
8.1.11. 4-Amino-2-(benzylsulfanyl)-5-(trifluoromethyl)-
5,6-dihydrofuro[2,3-d]pyrimidin-5-ol 10. 6-Amino-2-
(benzylsulfanyl)-4(3H)-pyrimidinone 9a (1 g, 4.3 mmol)
and bromotrifluoroacetone 224 (1 g, 5.2 mmol) were
suspended in ethanol (30 mL) and stirred under nitrogen
at 60 8C for 17 h. The solvent was evaporated under reduced

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
953
pressure and the residue was dissolved in ethyl acetate, and
purified by silica gel column chromatography (1:2 solution
of ethyl acetate/n-hexane) and then recrystallised from
diethyl ether/n-hexane. The product 10 was obtained as a
white crystalline solid (0.6 g, 1.7 mmol, 40%; mp 145–
147 8C). Found: HRMS (EI) 343.0615, C14H12F3N3O2S
requires 343.0602. dH (DMSO) 4.31 (2H, s, C(8)H2), 4.33–
4.39 (1H, m, 1£C(6)H2), 4.74–4.77 (1H, m, 1£C(6)H2),
6.35 (1H, br s, OH), 7.18–7.46 (5H, m, 2£C(10)H,
2£C(11)H, C(12)H). dC (DMSO) 34.35 (C-8), 76.12
(C-6), 79.2 (C-13, J¼48.2 Hz), 88.42 (C-5), 127.33
(C-4a), 127.35 (C-12), 128.74 (2£C-10), 129.37 (2£C-11),
138.62 (C-9), 160.28 (C-4), 172.46 (C-7a), 174.28 (C-2).
(2H, s, C(8)H2), 7.04 (1H, s, C(5)H), 7.19–7.51 (5H, m,
2£C(10)H, 2£C(11)H, C(12)H), 12.29 (1H, s, N(7)H),
12.66 (1H, s, N(3)H). dC (DMSO) 14.63 (C-15), 34.01
(C-8), 60.71 (C-14), 105.95 (C-4a), 109.53 (C-5), 122.85
(C-6), 127.68 (C-12), 128.79 (2£C-10), 129.71 (2£C-11),
137.71 (C-9), 149.83 (C-7a), 157.29 (C-4), 159.05 (C-2),
160.68 (C-13). nmax (KBr) 3486 (NH), 1717 (CvO), 1624,
1495, 1241, 698 cm21
.
8.1.14. 2-Amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile 13. To 2-amino-4-oxo-4,7-dihydro-3H-pyr-
rolo[2,3-d]pyrimidine-5-carbonitrile 136 (1.0 g, 5.7 mmol)
was added phosphoryl chloride (10 mL, 70 mmol) and N,N-
diisopropylamine (1.0 g, 10 mmol) and the solution was
heated to reflux overnight. At the conclusion of this period
the mixture was poured in cold-ice water (100 mL) and
stirred for 1 h, whereupon, dichloromethane (50 mL) was
added. This mixture was filtered and the filtrate was washed
with methanol, and ether to afford the crude product.
Purification by column chromatography on silica gel (ethyl
acetate/methanol—4/1) afforded the title compound 13 as
an amorphous solid (0.3 g, 1.55 mmol, 27%; mp .260 8C
(lit.20 .300 8C)). Found: HRMS (EI) found 193.0139,
195.0124. C7H4N355–37Cl requires 193.0155, 195.0126. dH
(DMSO) 6.91 (2H, br s, N(8)H2), 8.10 (1H, s, C(6)H), 12.5
(1H, s, N(3)H). dC (DMSO) 83.5 (C-5), 106.5 (C-4a), 115.4
(C-9), 134.5 (C-6), 151.5, 155, 160.8 (C-2, C-7a, C-4).
n
max (KBr) 3508 and 3305 (NH2), 3159, 1645, 1606, 1481,
.
1183, 1172, 713 cm21
8.1.12. Ethyl (2Z)-3-[4-amino-2-(benzylsulfanyl)-6-oxo-
1,6-dihydro-5-pyrimidinyl]-2-(hydroxyimino)-propano-
ate 11. 6-Amino-2-(benzylsulfanyl)-4(3H)-pyrimidinone 9a
(1 g, 4.31 mmol) was dissolved in dry DMF (15 mL).
Triethylamine (0.43 g, 4.31 mmol) was added and the
mixture was stirred under nitrogen at room temperature. A
solution of the ethyl 3-bromopyruvate oxime (1 g,
4.76 mmol) in dry DMF (15 mL) was added over a period
of 5 h to the stirred solution with the aid of a syringe pump.
Stirring was continued for a further hour after the addition.
The solution was evaporated under reduced pressure and the
residue was dissolved in ethyl acetate/methanol (4:1
solution, 10 mL). The resulting solution was absorbed on
top of a silica gel chromatography column and it was eluted
with 100% ethyl acetate, increasing the polarity up to 4:1
ethyl acetate/methanol solution. The product 11 was
obtained as a pale yellow solid (0.69 g, 1.90 mmol, 44%;
mp 184–186 8C (dec)). Found: HRMS (FAB) 363.1131,
C16H19N4O4S (Mþ1) requires 363.1127. dH (DMSO) 1.17
(3H, t, J¼7.1 Hz, C(18)H3), 3.44 (2H, s, C(13)H2), 4.10
(2H, q, J¼7.1 Hz, C(17)H2), 4.33 (2H, s, C(8)H2), 6.25 (2H,
br s, NH2), 7.22–7.32 (3H, m, 2£C(11)H, C(12)H), 7.42–
7.43 (2H, m, 2£C(10)H), 11.66 (1H, br s, NOH), 12.16 (1H,
br s, N(1)H). dC (DMSO) 14.28 (C-18), 19.67 (C-13), 33.55
(C-8), 60.92 (C-17), 88.56 (C-5), 127.55 (C-12), 128.78
(2£C-11), 129.51 (2£C-10), 138.23 (C-9), 150.66 (C-14),
160.24, 163.05, 163.13, 164.10 (C-2, C-4, C-6, C-15). nmax
(KBr) 3498 (NH2), 3360 (NH), 1731 (CvO), 1575, 1422,
8.1.15. 2-Amino-4-[(4-chlorophenyl)sulfanyl]-7H-pyr-
rolo[2,3-d]pyrimidine-5-carbonitrile 14. Trifluoroacetic
anhydride (2.42 mL, 17 mmol) was added dropwise over a
period of 15 min to a stirred suspension of 2-amino-4-oxo-
4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile 3a
(1 g, 5.7 mmol) in dry pyridine (15 mL) at 0 8C (ice-water
bath) under a nitrogen atmosphere. After 20 min, solid
(4-chloro)thiophenol (2.061 g, 14.2 mmol) was added, and
the stirred reactants were allowed to warm up to room
temperature. After a further period of 2 h, conc. aqueous
ammonia (d 0.88, 5.7 mL) was added dropwise over a
period of 10 min, followed by 27% aqueous hydrogen
peroxide (0.57 mL). After the reaction mixture had been
stirred for a further period of 3 h, the products were
evaporated to dryness under reduced pressure. The residue
was re-evaporated with toluene (20 mL) under reduced
pressure, and was then shaken with toluene (15 mL) and
water (15 mL). The resulting mixture was filtered and the
residue washed first with toluene and then with water.
Column chromatography of the residue on silica gel with
ethyl acetate/acetone 1/1 gave the title compound 14 as an
amorphous, nearly colourless solid (0.90 g, 3 mmol, 52%;
mp .260 8C). Found: HRMS (EI) 301.0189, 303.0179
C13H835–37ClN5S requires 301.0189, 303.0159. dH (DMSO)
6.39 (2H, s, N(8)H2), 7.50–7.53 (2H, m, 2£C(11)H), 7.60–
7.64 (2H, m, 2£C(12)H), 8.00 (1H, s, C(6)H), 12.27 (1H, s,
N(7)H). dC (DMSO) 83.08 (C-5), 106.12 (C-4a), 116.43
(C-14), 126.92 (C-13), 129.61 (2£C-11), 133.11 (C-6),
134.44 (C-10), 136.76 (2£C-12), 152.98, 159.76, 160.55
(C-2, C-7a, C-4).
1235, 1128, 770, 705 cm21
.
8.1.13. Ethyl 2-(benzylsulfanyl)-4-oxo-4,7-dihydro-3H-
pyrrolo[2,3-d]pyrimidine-6-carboxylate 12. Ethyl (2Z)-
3-[4-amino-2-(benzylsulfanyl)-6-oxo-1,6-dihydro-5-pyri-
midinyl]-2-(hydroxyimino)propanoate 11 (0.5 g,
1.38 mmol) was suspended in a mixture of ethanol
(25 mL), water (25 mL) and conc. hydrochloric acid (5
drops). Benzaldehyde (4 mL) was added and the reaction
mixture was heated to reflux under nitrogen until reaction
was complete as shown by TLC. The solvent was removed
under reduced pressure using co-evaporation with toluene/
ethanol to remove residual water. The residue was
suspended in ethyl acetate/diethyl ether (1:1 solution,
20 mL) and filtered to afford the title compound 12 as an
off-white microcrystalline solid (0.37 g, 1.12 mmol, 81%;
mp 185 – 188 8C). Found: HRMS (EI) 329.0849,
C16H15N3O3S requires 329.0834. dH (DMSO) 1.31 (3H, t,
J¼7.1 Hz, C(15)H3), 4.29 (2H, q, J¼7.1 Hz, C(14)H2), 4.43
8.1.16. Synthesis of N-(7-acetyl-5-cyano-4-oxo-4,7-dihy-
dro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)acetamide 15. A
mixture of 2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile 3a (2 g, 11.4 mmol), acetic

954
C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
anhydride (30 mL) and a catalytic amount of DMAP were
heated to reflux for 6 h. The mixture was then evaporated
under reduced pressure to dryness and the residue was
washed with acetone and diethyl ether, to yield the title
compound 15 (2.406 g, 9.26 mmol, 81%; mp .240 8C).
Found: HRMS (FAB) 260.0765, C11H10N5O3 (Mþ1)
requires 260.0784. dH (DMSO) 2.21 (3H, s, C(10)H3),
2.84 (3H, s, C(13)H3), 8.34 (1H, s, C(6)H), 11.71 (1H, br s,
N(8)H), 12.11 (1H, br s, N(3)H). dC (DMSO) 24.33 (C-13),
25.79 (C-10), 90.40 (C-5), 105.10 (C-4a), 113.98 (C-11),
129.51 (C-6), 148.70, 149.17, 155.46 (C-2, C-4, C-7a),
168.31, 174.27 (C-9, C-12). nmax (KBr) 3450, 3136, 2230
(2H, br s, NH2), 6.18 (2H, br s, NH2), 7.29 (2H, d, J¼7.8 Hz,
2£C(8)H), 7.44 (2H, d, J¼7.8 Hz, 2£C(9)H). dC (DMSO)
21.71 (C-11), 90.05 (C-5), 126.49 (C-7), 131.26 (2£C-8),
136.36 (2£C-9), 140.16 (C-10), 163.28 (C-4), 164.96 (C-2),
170.12 (C-6) nmax (KBr) 3485, 3371, 1642, 1619, 1558,
1363, 899, 506 cm21
.
8.2.3. 7-(Benzylsulfanyl)imidazo[1,2-c]pyrimidin-5-
ylamine 21. To a suspension of 6-(benzylsulfanyl)-2,4-
pyrimidinediamine 20a (1 g, 4.31 mmol) and sodium
acetate (0.707 g, 8.62 mmol) in water (20 mL) was added
chloroacetaldehyde (50% in water) (0.744 g, 4.74 mmol).
This was left stirring at 50 8C overnight. After the solution
was evaporated to dryness, the residue was suspended in
10 mL of a 4:1 solution of ethyl acetate/methanol and
filtered. The solution was purified by silica gel chromato-
graphy, using 4:1 ethyl acetate/methanol as eluent. Evap-
oration of the relevant fractions afforded the title compound
21 as an off-white solid (0.276 g, 1.08 mmol, 25%; mp
.240 8C). Found: HRMS (FAB) 257.0851, C13H13N4S
(Mþ1) requires 257.0861. dH (DMSO) 4.32 (2H, s,
C(10)H2), 6.66 (1H, s, C(8)H), 7.21–7.24 (1H, m,
C(14)H), 7.28–7.35 (2H, m, 2£C(13)H), 7.39–7.44 (2H,
m, 2£C(12)H), 7.40 (1H, s, C(3)H), 7.73 (2H, br s, NH2),
7.79 (1H, s, C(2)H). dC (DMSO) 34.47 (C-10), 95.96 (C-8),
108.37 (C-3), 127.43 (C-14), 128.84 (2£C-13), 129.24
(2£C-12), 133.39 (C-2), 138.10 (C-11), 146.34 (C-7),
147.33 (C-5), 150.93 (C-8a). nmax (KBr) 3435 (NH2),
(CN), 1729 (CvO), 1707, 1658, 1371, 782 cm21
.
8.1.17. 7-Acetyl-2-amino-4-oxo-4,7-dihydro-3H-pyr-
rolo[2,3-d]pyrimidine-5-carbonitrile 16. A mixture of
2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile 3a (0.2 g, 1.13 mmol), acetic anhydride
(0.159 g, 1.2 mmol) and DMAP (5 mg, catalytic quantity)
was gently heated for 5 min with a heating gun. The mixture
was then evaporated under reduced pressure, filtered and
washed with acetone and diethyl ether and dried under
reduced pressure to give the title compound 16 as a
colourless solid (0.18 g, 0.83 mmol, 73%; .240 8C).
Found: HRMS (FAB) found 218.0669, C9H8N5O2 (Mþ1)
requires 218.0678. dH (DMSO) 2.81 (3H, s, C(11)H3), 6.78
(2H, br s, NH2), 8.03 (1H, s, C(6)H), 11.11 (1H, s, NH). dC
(DMSO) 25.77 (C-11), 90.51 (C-5), 100.26 (C-4a), 114.58
(C-9), 126.60 (C-6), 151.88, 154.75, 157.46 (C-2, C-4,
C-7a), 168.72 (C-10). nmax (KBr) 3418, 3324, 2228 (CN),
3111, 3081, 1670, 1546, 1310, 1156, 931, 742, 704 cm21
.
1742 (CvO), 1683, 1635, 1593, 1374, 1310, 780 cm21
.
8.2.4. 5-(Benzylsulfanyl)diimidazo[1,2-a:1,2-c]pyri-
midine 22. To a suspension of 6-(benzylsulfanyl)-2,4-
pyrimidinediamine 20a (0.5 g, 2.15 mmol) and sodium
acetate (0.350 g, 4.26 mmol) in water (20 mL) was added
chloroacetaldehyde (50% in water) (0.843 g, 5.37 mmol).
This was left stirring at 50 8C for 22 h. After the solution
was evaporated to dryness, the residue was suspended in
10 mL of a 5:1 solution of ethyl acetate/methanol and
filtered. The solution was purified by silica gel chromato-
graphy, using 100% ethyl acetate as eluent, and then 4:1
ethyl acetate/methanol. The product was obtained from
early fractions as a brown solid (0.144 g, 0.51 mmol, 24%;
mp 135–137 8C). Found: HRMS (FAB) 281.0863,
C15H13N4S (Mþ1) requires 281.0861. dH (DMSO) 4.48
(2H, s, C(12)H2), 7.21 (1H, s, C(6)H), 7.22–7.32 (3H, m,
C(16)H, 2£C(15)H), 7.32 (1H, s, C(3)H), 7.36–7.38 (2H,
m, 2£C(14)H), 7.55 (1H, s, C(8)H), 7.81 (1H, s, C(2)H),
8.10 (1H, s, C(9)H). dC (DMSO) 37.30 (C-12), 104.78
(C-6), 111.90 (C-2), 113.14 (C-9), 128.03 (C-16), 128.48
(C-3), 128.96 (2£C-15), 129.40 (2£C-14), 132.15 (C-13),
132.92 (C-8), 136.15 (C-10a), 136.89 (C-6a), 141.39 (C-5).
8.2. General method for the synthesis of benzylsulfanyl-
and (4-methylphenyl)sulfanyl-pyrimidines from
6-chloro-2,4-pyrimidinediamine
To a suspension of 6-chloro-2,4-pyrimidinediamine (1 g,
6.94 mmol) and sodium hydroxide (0.35 g, 8 mmol) in
ethanol (30 mL) and water (20 mL), the appropriate thiol
(1.3 g, 10.46 mmol) was added. The reaction mixture was
stirred at 80 8C overnight. The solution was concentrated by
evaporation under reduced pressure, and water (20 mL) was
added to the residue. The precipitate was collected by
filtration, washed with water and diethyl ether, to afford the
required product as a white solid.
8.2.1. 6-(benzylsulfanyl)-2,4-pyrimidinediamine 20a.
Obtained using benzylthiol in 85% yield (1.37 g,
5.90 mmol), mp 144–146 8C (lit.23 146–148 8C). Found:
HRMS (FAB) found: 233.0871, C11H13N4S (Mþ1) requires
233.0861. dH (DMSO) 4.26 (2H, s, C(7)H2), 5.63 (1H, s,
C(5)H), 6.01 (2H, s, NH2), 6.20 (2H, s, NH2), 7.20–7.40
(5H, m, 2£C(9)H, 2£C(10)H, C(11)H). dC (DMSO) 32.44
(C-7), 90.37 (C-5), 127.26 (C-11), 128.74 (2£C-9), 129.29
(2£C-10), 138.77 (C-8), 162.85 (C-4), 164.14 (C-2), 166.02
(C-6). nmax (KBr) 3440 (NH2), 3302, 1612, 1562, 1430,
n
max (KBr) 3125, 3041, 1628, 1568, 1311, 1135, 1030, 858,
.
707, 695 cm21
8.2.5. 5-Amino-7-(benzylsulfanyl)imidazo[1,2-c]pyri-
midine-3-carbonitrile 23. To a suspension of 6-(benzyl-
sulfanyl)-2,4-pyrimidinediamine 20a (0.3 g, 1.29 mmol)
and sodium acetate (0.212 g, 2.60 mmol) in water (15 mL)
was added freshly prepared chloro(formyl)acetonitrile
(0.147 g, 4.74 mmol). This was left stirring at 50 8C
overnight. After the solution was evaporated to dryness,
the residue was suspended in 10 mL of a 4:1 solution of
ethyl acetate/methanol and filtered. The solution was
1362, 787, 715 cm21
.
8.2.2. 6-[(4-Methylphenyl)sulfanyl]-2,4-pyrimidinedi-
amine 20b. Obtained using 4-methylbenzenethiol in 94%
yield (1.51 g, 6.50 mmol; mp 240–242 8C). Found: HRMS
(EI) found: 232.0788, C11H12N4S requires 232.0783. dH
(DMSO) 2.35 (3H, s, C(11)H3), 5.08 (1H, s, C(5)H), 5.97

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
955
purified by silica gel chromatography, using 4:1 ethyl
acetate/methanol as eluent. The title compound 23 was
obtained as a brown solid (0.105 g, 0.37 mmol, 29%; mp
.240 8C). Found: HRMS (FAB) 282.0819, C14H12N5S
(Mþ1) requires 282.0813. dH (DMSO) 4.45 (2H, s,
C(10)H2), 6.26 (1H, s, C(8)H), 7.22–7.44 (5H, m,
2£C(12)H2, 2£C(13)H2, C(14)H), 7.31 (2H, br s, NH2),
8.37 (1H, s, C(2)H). dC (DMSO) 33.23 (C-10), 89.77 (C-8),
92.12 (C-3), 113.42 (C-9), 127.53 (C-14), 128.83 (2£C-12),
129.31 (2£C-13), 137.90 (C-11), 146.26 (C-2), 149.62
(C-8a), 151.70 (C-5), 163.96 (C-7). nmax (KBr) 3340, 3122,
8.2.8. N 4-Allyl-6-(benzylsulfanyl)-2,4-pyrimidinedi-
amine 26. To a solution of 4-(benzylsulfanyl)-6-chloro-2-
pyrimidinamine 25 (2.6 g, 10.3 mmol) in methoxyethanol
(30 mL), was added allylamine (2.28 g, 3 mL, 40 mmol) at
room temperature. The resulting solution was stirred and
heated at 100 8C for 5 days. Then the solvent was
evaporated under reduced pressure and the residue was
purified by silica gel column chromatography using as
eluent a gradient of ethyl acetate/n-hexane (1:1)!100%
ethyl acetate. The product 26 was obtained as a white solid
(1.25 g, 4.6 mmol, 45%; mp 135–138 8C). Found: HRMS
(FAB) found: 273.1185, C14H17N4S (Mþ1) requires
273.1174. dH (DMSO) 3.84 (2H, br s, C(14)H2), 4.26 (2H,
s, C(8)H2), 5.05 (1H, dd, J¼10.3, 1.6 Hz, 1£C(16)H2), 5.14
(1H, dd, J¼17.2, 1.6 Hz, 1£C(16)H2), 5.66 (1H, s, C(5)H),
5.80–5.89 (1H, m, C(15)H), 6.05 (2H, br s, NH2), 6.83 (1H,
br s, N(13)H), 7.20–7.24 (1H, m, C(12)H), 7.27–7.31 (2H,
m, C(10)H2), 7.38–7.40 (2H, m, C(11)H2). dC (DMSO)
32.48 (C-8), 42.61 (C-14), 90.30 (C-5), 115.38 (C-16),
127.23 (C-12), 128.71 (2£C-10), 129.27 (2£C-11), 136.21
(C-9), 138.82 (C-15), 162.62, 163.05, 165.47 (C-2, C-4,
C-6). nmax (KBr) 3458, 3136, 1629, 1594, 1559, 1435, 1232,
2225 (CN), 1630, 1315, 705 cm21
.
8.2.6. Ethyl 5-amino-7-(benzylsulfanyl)imidazo[1,2-
c]pyrimidine-2-carboxylate 24. 6-(Benzylsulfanyl)-2,4-
pyrimidinediamine 20a (0.3 g, 1.29 mmol) was dissolved
in dry DMF (15 mL). Triethylamine (0.13 g, 1.29 mmol)
was added and the mixture was stirred under nitrogen at
room temperature. A solution of the ethyl 3-bromopyruvate
oxime (0.3 g, 1.42 mmol) in dry DMF (10 mL) was added at
room temperature over a period of 5 h to the stirred solution
with the aid of a syringe pump. TLC showed no reaction
after 2 h, so stirring was continued for a further 10 h at
80 8C. The solution was evaporated under reduced pressure
and the residue was dissolved in ethyl acetate/methanol (4:1
solution, 10 mL). The resulting solution was absorbed on
top of a silica gel chromatography column and it was eluted
with 100% ethyl acetate, increasing the polarity up to 4:1
ethyl acetate/methanol solution. The product 24 was
obtained as a brownish solid (0.07 g, 0.21 mmol, 16%; mp
.240 8C). HRMS (FAB) found: 329.1064, C16H17N4O2S
(Mþ1) requires 329.1072. dH (DMSO) 1.30 (3H, t, J¼7.1 Hz,
C(12)H3), 4.29 (2H, q, J¼7.1 Hz, C(11)H2), 4.33 (2H, s,
C(14)H2), 6.66 (1H, s, C(8)H), 7.22–7.25 (1H, m, C(18)H),
7.30–7.33 (2H, m, C(17)H2), 7.43–7.45 (2H, m, C(16)H2),
7.92 (2H, br s, NH2, exchange with D2O), 8.51 (1H, s, C(3)H).
dC (DMSO) 14.62 (C-12), 34.41 (C-14), 60.67 (C-11), 95.53
(C-8), 113.99 (C-3), 127.46 (C-18), 128.83 (2£C-16), 129.23
(2£C-17), 136.05 (C-15), 137.84 (C-2), 146.41 (C-8a), 147.42
(C-5), 153.11 (C-9), 162.79 (C-7). nmax (KBr) 3311, 3026,
1166, 788 cm21
.
8.2.9. N0-[4-(Allylamino)-6-(benzylsulfanyl)-2-pyri-
midinyl]-N,N-dimethylimidoformamide 27. To a stirring
suspension of N 4-allyl-6-(benzylsulfanyl)-2,4-pyrimidine-
diamine 26 (0.5 g, 1.84 mmol) in anhydrous DMF (5 mL)
was
added
bis(dimethylamino)-tert-butoxymethane
(Bredereck’s reagent) (0.38 g, 2.2 mmol) and the mixture
was heated to 60 8C under nitrogen for 20 min. The resulting
solution was concentrated under reduced pressure and the
residue was dissolved in ethyl acetate (5 mL). Upon
addition of diethyl ether a white product crushed out of
solution, which was filtered off and washed with diethyl
ether and dried under reduced pressure to afford the title
compound 27 as a white solid (0.526 g, 1.6 mmol, 87%; mp
133–135 8C). Found: HRMS (FAB) found: 328.1599,
C17H22N5S (Mþ1) requires 328.1596. dH (DMSO) 2.96
(3H, s, C(10)H3), 3.06 (3H, s, C(10a)H3), 3.88 (2H, br s,
C(12)H2), 4.32 (2H, s, C(16)H2), 5.06 (1H, dd, J¼10.3,
1.6 Hz, 1£C(14)H2), 5.15 (1H, dd, J¼17.2, 1.6 Hz,
1£C(14)H2), 5.86 (1H, m, C(13)H), 5.94 (1H, s, C(5)H),
7.04 (1H, t, J¼5.7 Hz, N(11)H), 7.21–7.24 (1H, m,
C(20)H), 7.28–7.32 (2H, m, 2£C(19)H), 7.38–7.39 (2H, m,
2£C(18)H), 8.53 (1H, s, C(8)H). dC (DMSO) 32.73 (C-16),
34.74 and 40.65 (C-10 and C-10a), 42.75 (C-12), 94.47 (C-5),
115.46(C-14), 127.24(C-20), 128.74(2£C-18), 129.11(2£C-
19),136.16(C-13), 138.67(C-17), 158.26(C-8),163.15(C-2),
165.68 (C-4). nmax (KBr) 3436, 3216, 1625, 1592, 1514, 1344,
1716 (CvO), 1626, 1530, 1341, 1014, 787 cm21
.
8.2.7. 4-(Benzylsulfanyl)-6-chloro-2-pyrimidinamine 25.
To a suspension of 4,6-dichloro-2-pyrimidinamine (2 g,
12.2 mmol) in 20 mL of ethanol, was added sodium
hydroxide (0.48 g, 12.2 mmol) in water (10 mL) with
stirring. After 5 min, benzylthiol (1.51 g, 12.2 mmol) was
added to the solution and almost immediately a white
precipitate formed. Then, water (20 mL) was added to the
solution and it was left stirring at room temperature for a
further 10 min. The product was collected by filtration,
washing extensively with water and diethyl ether, and dried
under reduced pressure affording the title compound 25 as a
white crystalline solid (2.8 g, 11.06 mmol, 91%; mp 118–
120 8C). Found: HRMS (FAB) 252.0367, C11H11N3S35Cl
(Mþ1) requires 252.0362; found 254.0315, C11H11N3S37Cl
(Mþ1) requires 254.0333. dH (DMSO) 4.39 (2H, s, C(8)H2),
6.60 (1H, s, C(5)H), 7.20 (2H, s, NH2), 7.22–7.43 (5H, m,
C(12)H, 2£C(11)H, 2£C(10)H). dC (DMSO) 32.80 (C-8),
105.18 (C-5), 127.56 (C-12), 128.84 (2£C-11), 129.42
(2£C-10), 137.77 (C-9), 159.36 (C-6), 162.63 (C-2), 171.45
(C-4). nmax (KBr) 3478, 3296, 1633, 1521, 1411, 1215, 816,
1111, 796, 713 cm21
.
8.2.10. N0-[1-Allyl-7-(benzylsulfanyl)-2-hydroxy-
1H,2H,3H-imidazo[1,2-c]pyrimidin-4-ium-5-yl]-N,N-
dimethylimidoformamide chloride 28. To a suspension of
N0-[4-(allylamino)-6-(benzylsulfanyl)-2-pyrimidinyl]-N,N-
dimethylimidoformamide 27 (0.25 g, 0.76 mmol) and
sodium acetate (0.125 g, 1.53 mmol) in acetonitrile
(10 mL) and water (3 mL) was added chloroacetaldehyde
(50% in water) (0.144 g, 0.91 mmol). This was left stirring
at 50 8C overnight. After the solution was evaporated to
dryness, the residue was taken in 20 mL of dichloro-
methane, washed with water (10 mL) and purified by silica
785, 696 cm21
.

956
C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
gel column chromatography (100% dichloromethane!
dichloromethane/methanol 9:1). Evaporation of the relevant
fractions afforded the title compound 28 as its chloride salt,
which was recrystallised from acetone/n-hexane as a white
crystalline solid (0.144 g, 0.35 mmol, 47%; mp 158–
160 8C). Found: HRMS (FAB) found: 370.1706,
C19H24N5OS (Mþ) requires 370.1702. dH (DMSO) 3.17
and 3.32 (2£3H, 2£s, C(15)H3, C(16)H3), 4.01–4.12 (2H,
m, C(9)H2), 4.18–4.23 (1H, m, 1£C(3)H2), 4.34–4.39 (1H,
m, 1£C(3)H2), 4.52 (2H, s, C(17)H2), 5.21 (1H, d, J¼10.3,
1.1 Hz, 1£C(11)H2), 5.38 (1H, dd, J¼17.2, 1.1 Hz,
1£C(11)H2), 5.52–5.57 (1H, m, C(2)H), 5.78–5.87 (1H,
m, C(10)H), 6.52 (1H, s, C(8)H), 7.22–7.38 (5H, m,
2£C(19)H, 2£C(20)H, C(21)H), 7.49–7.51 (1H, m, OH),
8.89 (1H, s, C(13)H). dC (DMSO) 34.00 (C-17), 35.91 and
41.98 (C-15, C-16), 44.41 (C-9), 53.12 (C-3), 82.07 (C-2),
88.11 (C-8), 118.76 (C-11), 127.63 (C-21), 128.90 (2£C-
19), 129.20 (2£C-20), 131.86 (C-10), 137.58 (C-18), 154.03
(C-8a), 154.68 (C-5), 160.02 (C-13), 173.59 (C-7). nmax (KBr)
rolo[2,3-d]pyrimidine-5-carbonitrile 30b. To 2-amino-4-
oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
3a (0.7 g, 3.97 mmol) and 1-O-acetyl-2,3,5-tri-O-benzoyl-b-
D-ribofuranose (2 g, 3.97 mmol) in acetonitrile (50 mL) were
added hexamethyldisilazane (0.62 mL, 3 mmol), trimethyl-
chlorosilane (0.4 mL, 3 mmol) and finally SnCl4 (0.6 mL,
5 mmol) in acetonitrile (10 mL). After a short period of
magnetic stirring everything had dissolved and the mixture
was stirred for 24 h at room temperature, then dichloro-
methane (75 mL) was added and the mixture was extracted
with satd NaHCO3 solution. After re-extracting the aqueous
phase with dichloromethane, the combined organic phase was
washed with satd NaCl solution, dried (Na2SO4) and
evaporated. The residue was dissolved in dichloromethane
and absorbed onto the top of a silica gel chromatography
column and eluted with dichloromethane (200 mL), then with
dichloromethane/methanol—98/2 to give a mixture of two
inseparable anomers 30a and 30b as above (0.50 g,
0.81 mmol, 20%). Found: HRMS (FAB) found 620.1766,
C33H26N5O8 (Mþ1) requires 620.1781. dH (CDCl3) 4.48–
4.87 (3H, m, C(50)H2, C(40)H), 5.97–6.12 (3H, m, C(30)H,
C(20)H, C(10)H), 7.22–7.34 (9H, m, 6£C(90)H, C(6)H, NH2),
7.37–7.48 (3H, m, 3£C(100)H), 7.84–7.95 (6H, m,
3£C(80)H), 10.18 and 10.79 (1H, br s, N(3)H of a and b).
dC(CDCl3) 64.15 (C-50), 71.77, 74.31, 78.77 (C-20, C-30,
C-40), 86.18 (C-10), 870.77 (C-5), 100.85 (C-4a), 115.46 (CN),
128.54–128.61 (C-9 ), 128.97–129.43 0 (C-70), 129.84–
130.03 (C-80, C-6), 133.44–133.71 (C-10 ), 150.70, 151.09,
159.16 (C-2, C-7a, C-4), 165.27–166.80 (C-60). nmax (KBr)
3307 (NH2), 2228 (CN), 1725 (CvO), 1681, 1601, 1492,
3435, 3025, 1626, 1541, 1507, 1262, 1132, 1100, 947 cm21
.
8.2.11. 2-Amino-7-(20,30,50-tri-O-benzoyl-a-D-ribo-
furanosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one 29a and 2-amino-7-(20,30,50-tri-O-benzoyl-b-D-ribo-
furanosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one 29b. To 2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyri-
midin-4-one 3d (0.3 g, 1.98 mmol) and 1-O-acetyl-2,3,5-tri-
O-benzoyl-b-D-ribofuranose (1 g, 1.98 mmol) in 50 mL
acetonitrile were added hexamethyldisilazane (0.31 mL,
1.5 mmol), trimethylchlorosilane (0.2 mL, 1.5 mmol) and
finally stannic chloride (0.3 mL, 2.5 mmol) in acetonitrile
(10 mL) under nitrogen. After a short period of magnetic
stirring everything had dissolved and the mixture was stirred
for 21 h at room temperature, then dichloromethane
(75 mL) was added and the mixture was extracted with
aqueous satd NaHCO3 solution. After re-extracting the
aqueous phase with dichloromethane, the combined organic
phase was washed with satd NaCl solution, dried (Na2SO4)
and evaporated. The residue was dissolved in dichloro-
methane and absorbed into the top of a silica gel
chromatography column and eluted with dichloromethane
(200 mL), then with dichloromethane/methanol—98/2 to
give a mixture of two inseparable anomers 29a and 29b
(unknown ratio) as above (0.40 g, 0.673 mmol, 34%).
Found: HRMS (FAB) found 595.1814, C32H27N4O8
(Mþ1) requires 595.1829. dH(CDCl3) 4.58–4.75 (3H, m,
C(50)H2, C(40)H), 5.90–6.12 (3H, m, C(30)H, C(20)H,
C(10)H), 6.35 (1H, br s, C(5)H), 6.67 (1H, br s, C(6)H),
7.17–7.54 (11H, m, 6£C(90)H, 3£C(100)H, NH2), 7.73–
8.14 (6H, m, 6£C(80)H), 11.20 (1H, br s, N(3)H). dC(CDCl3)
64.34, 64.65 (C-50), 70.11, 72.00, 72.91, 74.23, 79.13,
80.25, 81.42, 85.22 (C-10, C-20, C-30, C-40), 101.57, 101.77
(C-40a), 103.24 (C-5), 118.11, 118.20 (C-6), 128.54–128.80
(C-9 ), 128.87–129.74 (C-70), 129.86–130.26 (C-80),
133.34–133.85 (C-100), 150.13, 150.84 (C-7a), 150.507,
151.32 (C-2), 161.09, 161.89 (C-4), 165.31–166.44 (C-6 ).
1271, 1123, 710 cm21
.
8.2.13. 2-Amino-6-{[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyl]amino}-4(3H)-pyrimidinone 33. To a suspen-
sion of 2-amino-6-chloropyrimidin-4(3H)-one (4 g,
27 mmol) in methoxyethanol (80 mL), was added triethyl-
amine (2.7 g, 27 mmol) and (2,2-dimethyl-1,3-dioxolan-4-
yl)methylamine (5 g, 38 mmol) with stirring. The mixture
was heated at reflux for 2 days, then the solvent was
evaporated under reduced pressure and ethyl acetate
(50 mL) was added. The resulting solution was left standing
overnight at 4 8C and a pale yellow precipitate formed,
which was filtered and washed with little ethyl acetate and
diethyl ether and dried under reduced pressure to afford the
title compound 33 as colourless microcrystals (4 g,
16 mmol, 62%; mp 134–136 8C). Found: HRMS (FAB)
found: 241.1301, C10H17N4O3 (Mþ1) requires 241.1301.
dH (DMSO) 1.24 and 1.32 (2£3H, s, 2£C(13)H3), 3.17–3.40
(2H, m, C(10)H2), 3.63 (1H, dd, J¼8.2, 6.1 Hz, C(8)H), 3.95
(1H, dd, J¼8.2, 6.1 Hz, C(8)H), 4.10–4.16 (1H, m, C(9)H),
4.49 (1H, s, C(5)H), 6.23 (2H, br s, NH2), 6.41 (1H, br s,
N(7)H), 9.77 (1H, br s, N(3)H). dC (DMSO) 25.71 and 27.21
(2£C-13), 41.93 (C-8), 67.17 (C-10), 72.52 (C-9), 74.6 (C-5),
108.73 (C-12), 155.47 (C-6), 163.18 (C-4), 164.53 (C-2). nmax
(KBr) 3351 (NH2), 2931, 1639, 1213, 787 cm21
.
n
max(KBr) 3368 (NH2), 2965 (CH), 1728, (CvO), 1671,
.
8.2.14. 2-Amino-7-[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyl]-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d] pyrimi-
dine-5-carbonitrile 34a and 2-amino-7-(2,3-dihydroxy-
propyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
dine-5-carbonitrile 34b. To a suspension of 2-amino-6-
{[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]amino}-4(3H)-
pyrimidinone 33 (1.5 g, 6.25 mmol) and sodium acetate
1607, 1580, 1455, 1269, 1125, 712 cm21
8.2.12. 2-Amino-7-(20,30,50-tri-O-benzoyl-a-D-ribo-
furanosyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
dine-5-carbonitrile 30a and 2-amino-7-(20,30,50-tri-O-
benzoyl-b-D-ribofuranosyl)-4-oxo-4,7-dihydro-3H-pyr-

C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
957
(1.2 g, 14.6 mmol) in water (28 mL), was added freshly
prepared chloro(formyl)acetonitrile (1.93 g, 18.75 mmol).
The mixture was left stirring for 20 h at 50 8C. A precipitate
was collected by filtration, washed with water, methanol and
diethyl ether to give a 2.5:1 mixture (by NMR) of 34a and
34b, respectively (0.995 g, 3.7 mmol, 60%). Found:(FAB)
a: 290.1263, C13H16N5O3 (Mþ1) requires 290.1253. dH
(DMSO) 1.23 and 1.31 (2£3H, 2£s, 2£C(13)H3 of a), 3.25–
3.38 (2H, m, C(10)H2 of b), 3.71–3.74 (2H, m, C(8)H2 of
b), 3.79–3.84 (2H, m, C(8)H2 of a), 3.95–3.99 (2H, m,
C(10)H2 of a), 4.08–4.12 (1H, m, C(9)H of b), 4.35–4.41
(1H, m, C(9)H of a), 4.74 (1H, t, J¼5.5 Hz, C(10)H2OH of
b), 5.00 (1H, d, J¼5.2 Hz, C(9)HOH of b), 6.50 (2H, s,
N(2)H2 of b), 6.52 (2H, s, N(2)H2 of a), 7.59 (1H, s, C(6)H
of b), 7.65 (1H, s, C(6)H of a), 10.72 (1H, s, N(3)H of b),
10.75 (1H, s, N(3)H of a). dC (DMSO) 25.47 and 26.95
(2£C-13 of a), 47.37 (C-8 of a), 48.23 (C-8 of b), 63.89
(C-10 of b), 66.40 (C-10 of a), 70.16 (C-9 of b), 77.93 (C-9
of a), 84.77 (C-11 of b), 85.27 (C-11 of a), 99.48 (C-4a of
b), 99.99 (C-4a of a), 109.36 (C-12 of a), 115.85 (C-5 of a),
116.05 (C-5 of b), 131.32 (C-6 of a), 131.77 (C-6 of b),
151.22 (C-7a of b), 151.32 (C-7a of a), 154.06 (C-2 of b),
154.23 (C-2 of a), 157.73 (C-4 of a), 157.76 (C-4 of b).
The resulting residue was taken in water (10 mL), filtered,
washed with ethanol and water, and dried under reduced
pressure to afford the required ester 31 as an amorphous, off
white solid (0.44 g, 1.56 mmol, 92%; mp .240 8C). Found:
HRMS (FAB) found: 283.1045, C11H15N4O5 (Mþ1)
requires 283.1042. dH (DMSO) 3.26–3.37 (2H, m,
C(10)H2), 3.69 (3H, s, C(13)H3), 3.75 (1H, m, C(9)H),
3.86–3.91 (1H, m, C(8)H), 4.13–4.17 (1H, m, C(8)H), 7.05
(2H, br s, N(2)H2), 7.48 (1H, s, C(6)H). dC (DMSO) 48.24
(C-8), 51.33 (C-13), 63.76 (C-10), 70.46 (C-9), 97.90
(C-4a), 109.36 (C-5), 129.89 (C-6), 149.08 (C-7a), 153.14
(C-2), 156.89 (C-4), 163.46 (C-11). nmax (KBr) 3325 (NH2),
1735 (CvO), 1667, 1311, 1107, 1063, 754 cm21
.
8.2.17. Ethyl 3-(2-amino-4-{[(2,2-dimethyl-1,3-dioxolan-
4-yl)methyl]amino}-6-oxo-1,6-dihydro-5-pyrimidinyl)-
2-(hydroxyimino)propanoate 36. 2-Amino-6-{[(2,2-
dimethyl-1,3-dioxolan-4-yl)methyl]amino}-4(3H)-pyrimi-
dinone 33 (1 g, 4.16 mmol) was dissolved in dry DMF
(15 mL). Triethylamine (0.5 g, 5 mmol) was added and the
mixture was stirred under nitrogen at room temperature. A
solution of ethyl 3-bromopyruvate oxime (1 g, 4.76 mmol)
in dry DMF (8 mL) was added over a period of 5 h to the
stirred solution with the aid of a syringe pump. Stirring was
continued for a further hour after the addition. The solution
was evaporated under reduced pressure and the residue was
dissolved in water (20 mL). The resulting solution was
cooled to 4 8C and filtered to give the title compound 36 as a
white solid (0.6 g, 1.62 mmol, 39%; mp 233–237 8C).
Found: HRMS (FAB) found: 370.1739, C15H24N5O6
(Mþ1) requires 370.1727. dH (DMSO) 1.17 (3H, t,
J¼7.1 Hz, C(19)H3), 1.25 and 1.33 (2£3H, 2£s,
2£C(13)H3), 3.24–3.54 (2H, m, C(10)H2), 3.39 (2H, s,
C(14)H2), 3.63 (1H, dd, J¼8.2, 6.1 Hz, C(8)H), 3.93 (1H,
dd, J¼8.2, 6.1 Hz, C(8)H), 4.01–4.15 (1H, m, C(9)H), 4.10
(2H, q, J¼7.1 Hz, C(18)H2), 5.87–5.90 (1H, m, N(7)H),
6.18 (2H, s, N(2)H2), 9.93 (1H, br s, N(1)H), 12.16 (1H, br s,
NOH). dC (DMSO) 14.28 (C-19), 19.08 (C-14), 25.75 and
27.19 (2£C-13), 43.63 (C-8), 60.92 (C-18), 67.18 (C-10),
74.89 (C-9), 82.47 (C-5), 108.66 (C-12), 151.71 (C-4),
153.87 (C-2), 161.0 (C-15), 162.13 (C-6), 164.37 (C-16).
nmax (KBr) 3425, 3335, 1702 (CvO), 1593, 1483, 1326,
8.2.15. 2-Amino-7-[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyl]-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi-
dine-5-carboxylic acid 35a and 2-amino-7-(2,3-
dihydroxypropyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-5-carboxylic acid 35b. The obtained 2.5:1
mixture of nitriles 203 and 204 (0.900 g, 3.3 mmol) was
dissolved in 5 M aqueous potassium hydroxide (15 mL).
The solution was heated to reflux for 5 h. After cooling, the
mixture was neutralised with glacial acetic acid and cooled
to 5 8C. The precipitate was filtered off, washed with water,
ethanol, and diethyl ether, to give the required carboxylic
acid as a 1:3 mixture (by NMR) of 35a and b, respectively
(0.920 g, 3.2 mmol, 97%). Found: HRMS (FAB) found for
b: 269.0861, C10H13N4O5 (Mþ1) requires 269.0885; for a:
309.1217, C13H17N4O5 (Mþ1) requires 309.1199. dH
(DMSO) 1.24 and 1.32 (2£3H, 2£s, 2£C(13)H3 of a),
3.26–3.38 (2H, m, C(10)H2 of b), 3.71–3.88 (2H, m,
C(8)H2 of b), 3.72–3.76 (2H, m, C(8)H2 of a), 3.97–4.00
(2H, m, C(10)H2 of a), 4.09–4.17 (1H, m, C(9)H of b),
4.40–4.43 (1H, m, C(9)H of a), 4.77 (1H, br s, C(10)H2OH
of b), 5.02 (1H, br s, C(9)HOH of b), 6.75 (2H, s, N(2)H2 of
a and b), 7.48 (1H, s, C(6)H of b), 7.52 (1H, s, C(6)H of a),
11.75 (1H, br s, N(3)H of a and b), 14.36 (1H, br s,
C(14)OOH of a and C(11)OOH of b). dC (DMSO) 25.50
and 26.97 (2£C-13 of a), 47.15 (C-8 of a), 47.91 (C-8 of b),
63.89 (C-10 of b), 66.47 (C-10 of a), 70.34 (C-9 of b), 74.13
(C-9 of a), 96.69 (C-4a of b and a), 109.39 (C-12 of a),
109.71 (C-5 of b), 110.21 (C-5 of a), 129.39 (C-6 of a),
130.03 (C-6 of b), 152.09 (C-7a of b), 152.22 (C-7a of a),
154.26 (C-2 of b), 154.62 (C-2 of a),162.48 (C-4 of b), 162.
63 (C-4 of a), 163.65 (C-14 of a, and C-11 of b). nmax (KBr)
827, 778, 701 cm21
.
8.2.18. Ethyl 2-amino-7-(2,3-dihydroxypropyl)-4-oxo-
4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxylate
32. Ethyl 3-(2-amino-4-{[(2,2-dimethyl-1,3-dioxolan-4-
yl)methyl]amino}-6-oxo-1,6-dihydro-5-pyrimidinyl)-2-
(hydroxyimino)propanoate 36 (0.35 g, 0.95 mmol) was
suspended in a mixture of ethanol (50 mL), water (15 mL)
and conc. hydrochloric acid (6 drops). Benzaldehyde (4 mL)
was added and the reaction mixture was heated under reflux
under nitrogen for 2 days. The solvent was removed under
reduced pressure using co-evaporation with toluene to
remove residual water. The residue was recrystallised from
ethanol–water to give the title compound 32 as a white solid
(0.18 g, 0.61 mmol, 64%; mp .240 8C). Found: HRMS
(FAB) found: 297.1202, C12H17N4O5 (Mþ1) requires
297.1199. dH (DMSO) 1.28 (3H, t, J¼7.1 Hz, C(11)H3),
3.24–3.28 (2H, m, C(14)H2), 3.75–3.80 (1H, m, C(13)H),
4.21 (2H, q, J¼7.1 Hz, C(10)H2), 4.25–4.38 (2H, m,
C(12)H2), 4.53 (1H, t, J¼5.7 Hz, C(14)H2OH), 4.67 (1H,
d, J¼5.1 Hz, C(13)HOH), 6.58 (2H, br s, N(2)H2), 7.04 (1H,
3436, 3349, 1688, 1646, 1423, 1069, 757 cm21
.
8.2.16. Methyl 2-amino-7-(2,3-dihydroxypropyl)-4-oxo-
4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carboxylate
31. To a suspension of the obtained 7:3 mixture of 35a and
35b (0.5 g, 1.7 mmol) in methanol (20 mL) was added
thionyl chloride (3 mL, 4 mmol). The solution was left
stirring for 3 h at 50 8C and then the solvent was evaporated.

958
C. L. Gibson et al. / Tetrahedron 60 (2004) 943–959
s, C(5)H), 10.57 (1H, br s, N(3)H). dC (DMSO) 15.08
(C-11), 46.58 (C-12), 60.57 (C-10), 64.82 (C-14), 71.46
(C-13), 101.05 (C-4a), 112.51 (C-5), 121.59 (C-6), 154.43
(C-2), 154.85 (C-7a), 159.54 (C-4), 161.47 (C-8). nmax
(KBr) 3346 (NH2), 1698 (CvO), 1603, 1265, 1187, 1098,
inhibition by the test compounds was calculated using the
following formula:
% inhibition ¼ 100ðControl 2 TestÞ=Test;
752 cm21
.
where ‘Control’ is the rate of reaction in the absence of the
test compound and ‘Test’ is the rate of reaction in presence
of the test compound.
8.3. Screening for GTPCH inhibitory activity
GTPCH I was kindly supplied by Professor A. Bacher and
Dr N. Schramek, of the Technical University, Munich. The
enzyme was stored in vials in aqueous solution at 275 8C, at
a concentration of 1.6 mg/mL (65 nM/mL of monomer).
Individual vials were thawed before use, and centrifuged to
remove insoluble material.
Acknowledgements
This research was supported by an EU TMR Network
‘Pteridines’ EUFMRXCT980204.
The HPLC screening assay was automated using a
Shimadzu LC-10ADvp liquid chromatograph linked to a
SIL-10ADvp auto-injector and a Shimadzu SPD-M10Avp
diode array detector. The column used was a Develosil
5 mm RP Aqueous column, 250£2.0 ID, fitted with a C18
guard column, both from Phenomenex. The mobile phase
comprised an aqueous solution containing triethylamine
(1%), isopropanol (0.8%) and 85% phosphoric acid (0.3%),
as described by Bacher et al.24 For the assay, a series of vials
in the HPLC autosampler contained the test compounds,
each vial containing one of the test compounds (4 mL of a
100 mM DMSO solution), GTP (80 mL of a 0.5 mM
solution), Tris buffer (20 mL of a 1 M solution, pH 8.5),
KCl (20 mL of a 1 M solution) and triply distilled water
(16 mL), making a total volume in each vial of 140 mL. The
control vial used DMSO (4 mL) containing no test
compound. To each of the test vials in turn at time zero,
the autosampler added a solution of GTP cyclohydrolase I
(60 mL) with mixing, making a total reaction mixture of
200 mL. The final concentration of enzyme in this reaction
mixture was 20 mM of active site equivalent (one active site
per monomer). The final concentration of GTP in the
reaction mixture was 200 mM, thus giving a 10:1 molar ratio
of GTP to enzyme active site. Samples of each reaction
mixture were injected on to the column after 23, 38, 53 and
68 min reaction time, each chromatogram being run for
13.5 min. Integration of the peak centred at 5.4 min gave a
measure of the amount of 7,8-dihydroneopterin triphosphate
formed in the reaction.
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