Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Article abstract of DOI:10.1039/b413655a

Two 17-mer oligodeoxynucleotide-5′-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was ³′ G G T A G T T A T T C C T T C T T ⁵′. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH₂₎₃OPO(OH)- linker, to the 5′-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)- pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate 3 the linker was -(CH₂₎₅CONH(CH₂₎₆OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)- pteridinone 36 was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, ⁵′T G A C C A T C A A T A A G¹⁴ G A A G¹⁸ A A G²¹ C C C T T C A G C G G C C ³′ 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

Full text of DOI:10.1039/b413655a

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A R T I C L E
Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and
their use as intermediates for the preparation of oligonucleotide
conjugates designed to target photooxidative damage on
single-stranded DNA representing the bcr–abl chimeric gene
Conor W. Crean,a Russell Camier,a Mark Lawler,b Clarke Stevenson,c R. Jeremy H. Davies,c
Peter H. Boyle*a and John M. Kelly*a
a Chemistry Department and Centre for Chemical Synthesis and Chemical Biology, Trinity
College, University of Dublin, Dublin 2, Ireland. E-mail: jmkelly@tcd.ie; Tel: 00 353 1 6081947
b Department of Haematology and Institute of Molecular Medicine, Trinity College, and St.
James’ Hospital, Dublin 8, Ireland
c School of Biology and Biochemistry, Queen’s University, Belfast, UK BT9 7BL
Received 6th September 2004, Accepted 12th October 2004
First published as an Advance Article on the web 15th November 2004
Two 17-mer oligodeoxynucleotide-5ꢀ-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as
photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1
representing the chimeric bcr–abl gene that is implicated in the pathogenesis ofꢀchronic myeloid leukaemia (CML).
The base sequence in the 17-mer was 3ꢀ G G T A G T T A T T C C T T C T T 5 . In the first of these ODN conjugates
(2) the pterin was attached at its N3 atom, via a –(CH2)3OPO(OH)– linker, to the 5ꢀ-OH group of the ODN. Conjugate
2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite
methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an
unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system.
In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products
containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and
pyrimido[1,2-c]pteridine (27). In the second ODN conjugate 3 the linker was –(CH2)5CONH(CH2)6OPO(OH)– and
was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of
2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone 36 was condensed with the hexylamino-modified 17-mer.
Excitation of 36 with near UV light in the presence of the ꢀsingle-stranded target 34-mer, 5ꢀ T G A C C A T C A A T A
A G14 G A A G18 A A G21 C C C T T C A G C G G C C 3 1 caused oxidative damage at guanine bases, leading to
alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine
sites with a marked preference for cleavage at G14. In contrast, excitation of ODN–pteridine conjugate 2 in the
presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14
(in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at
G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1
photochemical electron transfer, is operative. Much lower yields were found with the ODN–pteridine conjugate 3,
perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.
for the photooxidation of guanine in DNA primarily by a Type
Introduction
1
I mechanism, although they may also produce O2.13,14 More
Compounds containing the pteridine ring system are ubiquitous
in nature, often playing an essential role in the biochemical
processes of both plants and animals, including humans, and
their chemistry has been widely explored.1,2 It has been known
for a long time that pteridines occur in photosensitive organs
of both vertebrates and invertebrates,3,4 and some of the less
well known biological functions of pteridines depend upon their
photochemical properties. For example, pteridines have a role
in the photoreception and metabolism of plants,5–8 and have
been shown to be involved in the light-gathering chromophore
of DNA photolyase.9–11 There are also several reports in the
literature of pteridines causing photochemical damage on DNA
or its nucleobases. Thus, Chahidi et al. showed that the
pterin chromophore was efficiently quenched by guanosine,
the observed formation of the semi-reduced pterin molecule
indicating that its excited state had oxidised guanine,3 and Ito
and Kawanishi investigated the photooxidation properties of
a number of pteridine derivatives targeted to single-stranded
and double-stranded DNA sequences.12 These and other results
showed that pteridines are excellent photosensitising molecules
recently Hirakawa et al. have shown that a pteridine produced
by photohydrolysis of methotrexate, a widely used pteridine
antineoplastic agent, induces guanine-specific damage in DNA
through a photo-induced electron transfer mechanism,15 and the
highly fluorescent nature of many pteridines led Hawkins et al.
to prepare a series of pteridine nucleoside analogues, which were
incorporated into nucleic acids for use as fluorescent probes.16
Lastly, Bannwarth and his co-workers have described the linking
of several types of pteridine molecules to oligonucleotides.17,18
In the past few years considerable interest has been evinced
in the targeting of chemical reactions to particular sequences in
nucleic acids, since such procedures may have applications both
as sensors and in therapy.19,20 One approach is to use modified
oligonucleotides which are complementary to a particular se-
quence in the target (e.g. anti-sense or anti-gene methodologies),
for it is found that formation of conjugates can enhance stability,
control delivery to the cell and/or direct a reactive chemical
species to the desired target.20,21 The selectivity of such reactions
can be further enhanced by using photochemical methods,
and a range of photosensitiser–oligonucleotide conjugates has
3 5 8 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
©

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Fig. 1 17-mer ODN–pteridine conjugate hybridised to the target 34-mer 1.
been reported.22,23 For example, we have already targeted a
sequence using a non-intercalating ruthenium complex coor-
dinated to a complementary oligonucleotide sequence.24 The
target oligonucleotide corresponded to a chimeric nucleic acid
sequence generated by the fusion of the breakpoint cluster region
gene (bcr) and the abelson proto-oncogene (abl) found in chronic
myeloid leukaemia cells. Binding of this ruthenium sensitiser
to the 5ꢀ-end of the complementary sequence with subsequent
visible light irradiation, led to photooxidation on the target
strand with remarkable specificity (at G21).
In order to extend this study to a different class of photosensi-
tisers we have investigated the preparation of pteridine-coupled
oligodeoxynucleotides, and have explored their use in targeting
a specific sequence in a single-stranded nucleic acid. The target
1 (see Fig. 1) was the same synthetic 34-mer oligonucleotide
used in our earlier ruthenium work.24 Its base sequence acted as
a model for the mRNA sequence transcribed from the bcr–abl
fusion gene, which is present on the Philadelphia chromosome in
CML cells. The 17-mer oligonucleotide attached to the pteridine
incorporated the complementary base sequence (Fig. 1). In
most of the pteridine–nucleotide conjugates prepared up to
now by other workers the pteridine has been joined to the
nucleotide via its 2-amino group, through a linker chain. In
the present work we describe the formation of a new resonance-
stabilised heterocyclic cation 8 incorporating the rare 2H,6H-
pyrimido[6,1-b][1,3]oxazine ring system, which offers a route
to pteridines carrying a side chain attached to N-3. We report
some new chemistry of these side chain pteridines, leading up
to the attachment of 6,7-diphenylpterin at its N-3 position, via
a linker to the 17-mer oligodeoxynucleotide. 6,7-Diphenylpterin
was chosen as the pteridine photosensitiser because it absorbs
strongly in the near UV range, thus allowing its efficient selective
excitation.
solution, followed by evaporation of the solvent, gave 6 as an un-
stable solid. This on dissolving in dry tetrahydrofuran containing
benzil gave 2-amino-4-(3-chloropropoxy)-6,7-diphenylpteridine
9, which could also be obtained by direct hydrogenolysis of
5 in tetrahydrofuran followed by addition of benzil, without
isolation of intermediate 6. The proton NMR spectrum of
a solution of 2,5,6-triamino-4-(3-chloropropoxy)pyrimidine 6
dissolved in trideuteroacetonitrile showed signals due to the
three methylene groups, together with broad overlapping signals
due to the three amino groups. When D2O was added to this
solution, however, thus making the medium more polar, a
reaction occurred that could be followed in the sample tube by
proton NMR. The new product 8 still showed three methylene
group signals in its proton NMR spectrum, but at chemical shifts
different from those in 6. Furthermore, an aqueous solution of
it contained chloride ions (precipated with silver chloride). It
is believed that with addition of water, the more polar medium
encourages an intramolecular cyclisation of 6 to give the highly
resonance-stabilized multidentate bicyclic cation 8. The 2H,6H-
pyrimido[6,1-b][1,3]oxazine ring system of 8 is rare, and only a
few examples of it are known,29,30,31 although some time ago
we described26 the formation of an analogous cation 7, which
incorporates the 1,3-oxazolo[2,3-c]pyrimidine ring system, and
Lipkin and Lovett have reported 18O mechanistic studies on
another related bicyclic cation.32,33 The UV spectrum of 8 was
very similar to that of the previously reported cation 7. No
product was detected corresponding to cyclisation of the x-
chlorine atom of the side-chain of 6 with the exocyclic 5-amino
nitrogen atom.
The resulting oligonucleotide–pteridine conjugate 2 was de-
signed to hybridise to the target 34-mer 1 so that the pteridine
moiety, acting as a photosensitiser, would be held close to
the target guanine residue at position 21. This is illustrated in
Fig. 1. The linker between the pteridine and the oligonucleotide
in 2 is only three carbon atoms long. Accordingly, a second
oligonucleotide–pteridine conjugate 3 was also prepared, incor-
porating a much longer-chain linker. This enabled us to study
the effect of the linker chain length on the specificity of targeting
to the DNA.
Cation 8 is susceptible to attack by nucleophiles at several
different positions in the molecule. An aqueous solution of it
was formed directly by hydrogenolysis of 5 with palladium and
hydrogen in water, and addition of potassium hydroxide and
benzil to this solution gave the desired 3-(3-hydroxypropyl)-6,7-
diphenylpterin 10 in 66% yield. A similar reaction with butane-
2,3-dione gave the 6,7-dimethylpterin 12 (74%). Interestingly,
reaction with the unsymmetrical 1,2-dicarbonyl compound,
pyruvaldehyde, proceeded with a high degree of regiospecificity,
and gave only one of the two possible regioisomers in 95%
purity, as shown by NMR. While the spectra do not permit an
unambiguous assignment of regioisomers, we take the product
to be the 7-methylpterin 13, since the 5-amino group in cation
8 should be much more nucleophilic than the 6-amino group,
which is involved in resonance delocalisation of the positive
charge (pyrimidine numbering). The 5-amino group would thus
be expected to react preferentially with the more electrophilic
aldehyde group of the pyruvaldehyde, leading to 13, rather
than its 6-methyl regioisomer. A solid sample of the resonance
stabilised cation 8 was prepared by hydrogenation of 5 in water
Results and discussion
N-3 side chain pteridines25
The key starting material for the synthesis of 2 was 2-amino-3-
(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone, 10. Prepa-
ration of 10 started from 5-amino-7-methylthiofurazano[3,4-
d]pyrimidine 4, which upon treatment with 3-chloropropanol
in presence of bromine25,26 gave 5-amino-7-(3-chloropropoxy)
furazano[3,4-d]pyrimidine 5 (see Scheme 1). The proton NMR
spectrum of 5 is notable for the low field signal of the –OCH2–
group at d 4.67, reflecting the known very low electron density
at position 7 of furazano[3,4-d]pyrimidines.26,27 The furazan
ring in furazano[3,4-d] pyrimidines is also known to be cleaved
hydrogenolytically,28 and hydrogenolysis of 5 occurred readily
with palladised charcoal and hydrogen. The subsequent fate of
the initially formed air-labile diaminopyrimidine 6 was found,
however, to depend critically on the reaction conditions. Thus
hydrogenolysis of 5 in either a methanol or tetrahydrofuran
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 8 9

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Scheme 1 (i) 3-Chloropropanol, Br2; (ii) H2, Pd in tetrahydrofuran or H2O; (iii) H2O; (iv) benzil, tetrahydrofuran; (v) benzil, KOH, H2O, MeOH,
40 ◦C; (vi) biacetyl; (vii) pyruvaldehyde; (viii) benzil, MeOH, NaOMe.
followed by evaporation of solvent. When this sample of 8 was
refluxed in methanol containing sodium and benzil the main
product obtained was pteridine 10, together with a small amount
of 14. No N3-(3-methoxypropyl) product 11 was obtained,
suggesting that the oxazine ring in 8 (or in the corresponding
oxazinopteridine) is attacked nucleophilically by methoxide ion
at position A rather that at position B. This conclusion is
supported by similar results reported earlier for the analogous
resonance stabilised ion 7.26 Addition of sodium and benzil to
a methanol solution of triaminopyrimidine 6 led to formation
of the same two pteridines, 10 and 14. Under these conditions
cyclisation of 6 to cation 8 is not so favorable, and the main
product obtained here was the 4-methoxypteridine 14, which
could have been formed by direct methoxide ion substitution of
the chloropropoxy side chain in either pyrimidine 6 or pteridine
9. Again no N3-(3-methoxypropyl) product 11 was isolated.
The structure of compound 10, which was required for
preparation of its oligonucleotide conjugate 2, follows from its
spectroscopic properties, and also from the conversion of it into a
variety of new derivatives, some of them of novel structure. These
chemical interactions are described below and are summarised
in Scheme 2. Treatment of 10 with triphenylphosphine in dry
tetrachloromethane containing tetrahydrofuran gave 66% of the
expected chloropropyl pterin 18. In contrast, if the reaction
was carried out in pure tetrachloromethane the main product
obtained was the pteridine phosphazene 15 (47%), together
with 18 (39%). Very little work has been reported on pteridine
phosphazenes, and the formation of 15 occurs here under very
mild conditions without any specific deprotonation steps. Its
proposed structure is consistent with its observed spectroscopic
and chemical properties, including proton NMR spectra which
showed the presence of five phenyl groups, 31P NMR spectra
which showed the presence of a phosphorus atom, and elemental
analysis and high resolution mass spectra which corresponded
to the required formula of C39H31ClN5OP. In contrast to the
extreme insolubility of most pteridines, phosphazene 15 is
readily soluble in dichloromethane and ethyl acetate. It is
stable in refluxing aqueous ethanol, but suffers cleavage of
the phosphazene group in refluxing ethanolic hydrochloric
acid, to give the chloropropyl pterin 18. Another pteridine
phosphazene 29, which is readily soluble in acetone and in
dichloromethane, was prepared in 60% yield by treatment of
the 2-amino-4-methoxy pteridine 14 with triphenylphosphine in
tetrahydrofuran and tetrachloromethane.
Treatment of 18 with sodium hydroxide in aqueous ethanol
led to a smooth intramolecular cyclisation to give 20, which
incorporates the little known pyrimido[2,1-b]pteridine ring
system. Although first described many years ago by Angier
and Curran34 very little has since been published on this ring
system.35,36 The same tricyclic compound 20 could also be
obtained from the 3-hydroxypropylpteridine 10, by conversion
of the latter to its 2-hydroxypropylamino isomer 17 using a
base catalysed Dimroth rearrangement, and then heating 17
with triphenylphosphine in tetrachloromethane. Acetylation of
20 with acetic anhydride in pyridine gave its acetyl derivative
24, which was identical with the product obtained from 18
by acetylation of the latter in acetic anhydride and pyridine
to give 21 followed by base catalysed cyclisation of 21 to 24.
Interestingly, acetylation of 18 in acetic anhydride at room
temperature led to the unusual 2-diacetylaminopteridine 25. An
attempt to obtain a phthalimido derivative of 25 by nucleophilic
substitution of its chlorine atom using potassium phthalimide,
led instead to intramolecular cyclisation, giving again 24. A
monophthalimido derivative of 18, namely 19, could be obtained
by treatment of 18 with phthalic anhydride, and it was found
to be possible to convert 19 into its diphthalimido derivative
16 using potassium phthalimide in hexamethylphosphoramide.
An effort to convert 16 into 2-amino-3-(3-aminopropyl)-6,7-
diphenylpterin by treatment of it with hydrazine (Gabriel syn-
thesis) led only to the tricyclic cyclised compound 20, however.
The phthalimido ring of 19 could be partially hydrolysed
in high yield to the phthalic acid derivative 22 using dilute
3 5 9 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1

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Scheme 2 (i) PPh3–CCl4; (ii) KOH–H2O–MeOH; (iii) PPh3–CCl4, tetrahydrofuran; (iv) HCl–EtOH; (v) potassium phthalimide–HMPA;(vi) phthalic
anhydride–pyridine; (vii) NaOH–H2O–EtOH; (viii) Ac2O–pyridine; (ix) NaOH–H2O–EtOH–CH2Cl2; (x) NaOH–H2O–tetrahydrofuran; (xi) Ac2O,
20 ◦C; (xii) MeOH, reflux.
sodium hydroxide in aqueous ethanol–dichloromethane. The
tricyclic compound 20 underwent ring opening when treated
with concentrated potassium hydroxide in aqueous methanol,
to give the unstable 3-(3-aminopropyl)lumazine 23. When
heated in methanol solution 23 gave another unstable product,
believed to be the tricyclic compound 2,3-diphenyl-5,8,9,10-
tetrahydropyrimido[1,2-c]pteridin-6-one 27. The pyrimido[1,2-
c]pteridine ring system in 27 is also rare.37 The new compound 27
was too unstable to allow isolation of it in analytically pure form.
Evidence for the proposed structure 27, however, comes from its
high resolution mass spectrum, and also from its UV spectra
in neutral acid and alkaline solutions, all of which correspond
closely to the UV spectra reported for a closely related pteridine
analog 28.38 The unstable 3-(3-aminopropyl)lumazine 23 was
characterised by conversion of it into its acetyl derivative 26.
chain. Compound 10 also incorporates a side chain terminating
in a hydroxyl group, which feature allowed attachment of
the pteridine moiety to an oligonucleotide via the side chain
“linker” (see Scheme 3). This attachment was achieved by first
protecting the 2-amino group of 10 as its formamidine, 30,
and then treating the latter with either 2-cyanoethyl-N,N,Nꢀ,Nꢀ-
tetraisopropylaminophosphorodiamidite and tetrazole, or with
chloro-2-cyanoethyl-N,N-diisopropylaminophosphine. The lat-
ter, more reactive, phosphitylating reagent gave the cleaner
reaction and offered the method of choice for the preparation
of phosphoramidite 31. While not very stable, 31 could be
purified by column chromatography on neutral alumina, and
the pure material could be used for further work for up
to a week after its preparation. It was characterised by 31P
and 1H NMR spectra, the former showing a single peak at
d 148.4, characteristic of trivalent phosphorus. Interestingly,
if compound 10 were allowed to react with chloro(N,N-
diisopropylamino)methoxyphosphine without prior protection
of the 2-amino group, the product isolated was 32, in which the
Pteridine–oligonucleotide conjugate (2)
Compound 10 incorporates a pteridine moiety, designed as
the photosensitiser for inducing a lesion in an adjacent DNA
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 9 1

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Scheme 3 (i) CH(OMe)2NMe2; (ii) NCCH2CH2O-P(NPri2)Cl; (iii) DNA synthesiser; (iv) MeOP(NPri2)Cl.
=
Scheme
4
(i) HO2C(CH2)5NH2; (ii) Pd-charcoal, H2; (iii) benzil; (iv) BF4−Me2N+ C(NMe2)ON(COCH2)2; (v) 17-mer-OLIGO-O5ꢀ-
P(O)(OH)O(CH2)6NH2.
phosphorus atom has undergone oxidation to the pentavalent
state. A similar oxidation was observed by Frier et al. in attempts
to form phosphoramidites of flavin derivatives.39 Compound
32 could be converted to its corresponding formamidine 33.
The phosphorus(V) oxidation state in 32 and 33 was easily
demonstrated by the high field chemical shift of the phosphorus
atoms in their 31P NMR spectra, at d 14.5 and d 22.4 respectively.
The pteridine phosphoramidite 31 was attached to the syn-
thetic oligonucleotide using an Applied Biosystems 391 auto-
mated DNA synthesiser. The routine deprotection steps during
this procedure also removed the formamidine protecting group.
The product was purified by a combination of precipitation,
electroelution and preparative PAGE techniques, and it was
characterised as 2 by PAGE and UV spectroscopy and by mass
spectrometry. A negative ion electrospray mass spectrum of the
product exhibited a series of peaks representing multiply charged
negative ions. Deconvolution of this series gave a molecular
weight of 5592.7 for the synthesised conjugate. Structure 2 has a
molecular weight of 5592.8. The purified compound 2 was used
in photosensitisation experiments with a 34-mer oligonucleotide
target.
the target oligonucleotide (see below). Finally, in the synthesis
of conjugate 3, the pteridine moiety was attached to the 17-
mer oligonucleotide using a succinimide–ester coupling method,
instead of a phosphoramidite method.
Synthesis of the 17-mer ODN–pteridine conjugate 3 is
outlined in Scheme 4. The 5-carboxypentylamino side chain
of pyrimidine 35 was introduced by nucleophilic substitution
of the 2-methylthio group in the nitrosopyrimidine 34, using
a modification of a procedure described by Sugimoto et al.40
Compound 35 was then converted into the diphenylpteridine 36
by reduction followed by condensation with benzil. The carboxyl
group of 36 was then activated as its succinimide ester 37 using
a method developed by Bannwarth et al.41,42 involving treatment
of 36 with o-(N-succinimidyl)-N,N,Nꢀ,Nꢀ-tetramethyluronium
tetrafluoroborate. The N-hydroxy-succinimide activated ester
37 was allowed to react with the aminohexyl-modified 17-mer
oligonucleotide, giving the required oligonucleotide pteridine
conjugate 3.
Photolysis of free pteridine 36 with single stranded ODN 1
In order to assess whether a pteridine–ODN conjugate can direct
photo-specific damage to guanine bases in a complementary
target in a fashion similar to that which we have observed
with ruthenium conjugates,24 we have studied the photoproducts
generated by the pteridine-conjugates 2 and 3 with a single-
stranded complementary 34-mer DNA 1 (see Fig. 1). Initially it
was important to assess the effectiveness of the free pteridine in
bringing about photosensitized damage on the 34-mer target
1, and control experiments were set up, involving the UV
Pteridine–oligonucleotide conjugate (3)
Conjugate 3 differs from 2 in two main respects. Firstly, the
linker chain is attached at position 2 of the pteridine ring
system instead of at position 3, and secondly, the linker chain
is 14 atoms long, as opposed to 3 atoms long in 2. These
structural differences are expected to affect the efficiency of the
photochemical reaction when the conjugates are hybridised with
3 5 9 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1

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irradiation (k >330 nm) of the 34-mer target in the presence of an
equimolar amount of 36. The sites of cleavage were identified by
comparing with Maxam and Gilbert sequencing experiments43
in which treatment of the target 34-mer oligonucleotide with
formic acid followed by hot piperidine cleaves the target at every
purine base. The results of the subsequent PAGE electrophoresis
experiments are given in Fig. 2.
one and its precursor 2-amino-5-[(2ꢀ-deoxy-b-D-erythro-pento-
furanosyl)-amino]-4H-imidazol-4-one,45 in double-stranded
DNA, the dominant product should be 8-oxo-7,8-dihydro-
guanine. Although this latter compound does not react further
in hot piperidine, it is itself very readily photo-oxidised yielding
products such as the above mentioned oxazolone and imida-
zolone, spiroiminodihydantoin, guanidinohydantoin deriva-
tives and oxaluric acid.46
Photolysis of ODN–pteridine conjugate 2 with single stranded
ODN 1
The possibility of ODN–pteridine conjugates 2 and 3 being used
for site selective photoreaction was explored by allowing them to
hybridise with their complementary target 34-mer 1 (by warming
to about 85 ◦C and allowing to cool slowly), exposing to UV
light (k >330 nm), and then examining by gel electrophoresis
after treatment with piperidine.
Initial experiments were carried out with conjugate 3. Irra-
diation for extended periods (>2 hours) formed alkali-labile
sites. These occurred exclusively at guanine residues, with most
cleavage at G18.47 Similar results were observed with conjugate
2, but in this case the yields were much higher and this system
was therefore studied in more detail. The phosphoimagery trace
(Fig. 4) revealed that cleavage of the 34-mer occurred only at
guanine bases, but the cleavage occurred preferentially at bases
different from those damaged by free sensitiser 36. The major
cleavage site is G18 which is in the double-stranded region
and probably close to the pteridine-sensitiser moiety if it has
intercalated into the base-pair. The next strongest band is that
due to cleavage at G21 and this is expected to be located close
to the pteridine photosensitiser especially if the photosensitiser
does not intercalate (see Fig. 1). Damage at G14 and G15 is also
found but the yield is much lower. As pteridines are known to
undergo photo-induced electron transfer with guanine, leading
to the guanine radical cation (Type 1 process),12 it is possible that
the resultant migration of the oxidised centre through the base-
stack from G18 of the double-stranded portion of the hybrid
could explain the extent of cleavage at G14 and G15.44
Fig. 2 Irradiation of 34-mer target 1 in presence of pteridine 36 in
10 mM phosphate–100 mM NaCl solution (pH 6.9). Lane 1: 7.5 min
UV irradiation without piperidine treatment. Lane 2: 7.5 min irradiation
followed by piperidine treatment. Lane 3: 34-mer treated with formic
acid followed by piperidine.
From lane 2 it is clear that when pteridine 36 was excited in the
presence of the target 34-mer 1 in aerated solution, subsequent
treatment with hot piperidine induced strand breaks specifically
at guanine bases in 1. In contrast, irradiation without subsequent
piperidine treatment resulted in no cleavage of 1 (lane 1). This is
consistent with pteridine 36 causing oxidative damage at guanine
sites on the 34-mer 1, but producing lesions which do not lead
to cleavage of the phosphodiester backbone of DNA until the
sample is heated under alkaline conditions. (Blank experiments
also confirmed that the unsensitised 34-mer 1 is stable under
these conditions.) An interesting feature noticeable in Fig. 2 is
that cleavage did not occur at all guanine sites equally, there
being a significant preference for cleavage at G14, which is the
5ꢀG of the G14–G15 doublet in the target. Such preferential
reaction has been recognized for Type 1 processes on double
stranded DNA and has been attributed to the fact that the
HOMO for GG doublets is localised on the 5ꢀG, making this base
the most easily oxidised site.43 The fact that this pattern occurred
for the single stranded 34-mer target 1 may be attributed to the
reaction occurring in a hairpinned structure, in which the G14–
G15 doublet is now in a double stranded region, as shown in
Fig. 3.
Fig. 4 Densitometry plot of phosphorimage of gel from 30 min irradia-
tion experiment of duplex between 34-mer target 1 and ODN–pteridine
conjugate 2 (1 : 10 stoichiometry).
In the above experiment with 34-mer 1 and pteridine conjugate
2, reaction on the single-stranded portion was weak, except for
at G21. An interesting effect, however, was observed when the
sequence of the 34-mer target 1 was changed to that of 38. This
change involved replacing G21 by a C and replacing C23 by a G,
in effect moving the original G21 two bases further away from
the double stranded region (see Fig. 5). When the the duplex
between ODN–pteridine sensitiser 2 and variant target 38 was
irradiated and then treated with piperidine, the cleavage pattern
(G18 >> G14 > G15) in the double-stranded region was closely
similar to that found with target 1, the original 34-mer target.
In the single-stranded region little or no cleavage at G23 was
observed. A poorly resolved strong band was found, however,
corresponding to cleavage at G32, which is also in the single-
stranded region although seemingly remote from the pteridine
photosensitising molecule (see Fig. 6A). As with our earlier
work using a ruthenium conjugate,24 this cleavage is believed
Fig. 3 The 34-mer target ODN 1 showing hairpinning, with G14 and
G15 in a double stranded region.
Our experiments do not allow us to identify the degraded
guanine product which leads to cleavage of the DNA strand by
alkali treatment. While it is known that Type I processes with
2ꢀ-deoxyguanosine yield the alkali-labile products 2,2-diamino-
4-[(2ꢀ-deoxy-b-D-erythro-pentofuranosyl)amino]-5(2H)-oxazol-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 9 3

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Fig. 5 34-mer Target 1, hairpinning variant 38 and non-hairpinning variant 39.
targetted photoactivable reagents have been assessed. The first
exploits an unusual resonance-stabilised cation 8, which we have
shown to be a particularly valuable synthetic intermediate. The
ODN–conjugate 2 synthesised from this precursor is effective
at targeting photo-oxidative damage to its complementary
sequence. Alkali treatment causes cleavage at guanine, the
pattern being consistent with a Type I process, where the excited
state of the diphenylpterin abstracts an electron from guanine,
following which the hole may migrate through the base-pair
stack of the double helix. In the other ODN–conjugate 3 the
oligonucleotide was attached via a linking chain to the 2-amino
group of the diphenylpterin. This conjugate, however, was found
to be much less active, probably due to the much greater length
of the linker chain compared to that in 2.
Experimental
Thin layer chromatography was carried out on Merck Kieselgel
60 F254 0.2 mm silica gel plates. Flash chromatography was
carried out on Merck Kieselgel 60 (mesh 230–400 ASTM) silica
gel. Tetrahydrofuran was dried over calcium chloride, distilled
from lithium aluminium hydride, and then re-distilled from
sodium wire and benzophenone. Melting points are uncorrected.
Infrared spectra were recorded in Nujol mulls on Perkin Elmer
298, 599 or 883 spectrophotometers. Ultraviolet spectra were
recorded on Pye Unicam PU 8800, Pye Unicam SP8-200
or Perkin Elmer 402 UV/visible spectrophotometers. Nuclear
magnetic resonance spectra were recorded on Bruker 80, 300
1
or 400 instruments. Chemical shifts dH and dC, of H and 13C
respectively, are reported in ppm relative to tetramethylsilane
as an internal standard. Chemical shifts of 31P are relative
to 85% H3PO4. Coupling constants are quoted in Hertz.
Elemental analyses were carried out at either the Microana-
lytical Laboratory, National University of Ireland, Dublin; or
the Microanalytical Laboratory, University College London.
Electron ionisation mass spectrometry was carried out by Dr
Peter Bladon and Mr Mohammed Imran at the Department of
Pure and Applied Chemistry, University of Strathclyde, using
an AEI (Kratos) M59 spectrometer, modified to the MS 902
specification. Electrospray mass spectrometry was carried out
using a Micromass electrospray TOF instrument.
Fig. 6 Densitometry plots from 30 min irradiation of ODN–pteridine
conjugate 2 with (A) variant 38 (hairpinning) and (B) variant 39
(non-hairpinning).
to be due to the hairpinning shown in Fig. 7, which brings
G32 into close proximity with the pteridine. In support of this
contention, when another variant 39 (designed so as to eliminate
the possibility of hairpinning of the type shown in Fig. 7) was
used instead of 38 the band corresponding to cleavage at G32 was
greatly reduced (Fig. 6B). These results are entirely consistent
with the main reaction of pteridine conjugate 2 taking place
when the photosensitising pteridine is intimately associated with
the double-stranded region, possibly by intercalation. Reaction
with guanines outside this segment in the single-stranded region
requires the pteridine and guanine to be in close proximity, as
would be required for a Type 1 process.
Pteridinone–oligodeoxynucleotide conjugate 2
The 17-mer oligonucleotide component of 2 was first assembled
(1 lmol scale) on an Applied Biosystems 391 automated
DNA synthesiser using standard phosphoramidite chemistry.
Following deprotection of the 5ꢀ-dimethoxytrityl group, a so-
lution comprising (3-(2-N,N-dimethylaminomethyleneamino-
3,4-dihydro-4-oxo-6,7-diphenyl-3-pteridinyl)propoxy)(2-cyano-
ethoxy)(N,N-diisopropylamino)phosphoramidite 31 (17 mg,
0.027 mmol) dissolved in dry acetonitrile (365 ll) was introduced
onto the synthesis column. The coupling reaction was allowed
to proceed for 120 s and the synthetic cycle then completed.
The oligonucleotide conjugate thus formed was cleaved from
the solid support with concentrated ammonia and the solution
further heated at 55 ◦C for 3 h to remove base protecting
groups. After evaporation, the residual product was purified
by electrophoresis on 12% polyacrylamide gels containing
7 M urea. Bands corresponding to the pteridinone conjugate
were detected by UV shadowing, excised, and transferred to a
Biotrap electroelution apparatus (Schleicher and Schuell). After
running for 2 h at 2–3 W, the purified conjugate 2 was recovered
Fig. 7 Interaction of pteridine–ODN-duplex, with hairpinned variant
38.
Conclusion
Two approaches to the functionalisation of 6,7-diphenylpterins
and their conjugation to oligonucleotides to generate sequence-
3 5 9 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1

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from the anodic well in TRIS–borate buffer. It was precipitated
twice from sodium acetate solution with cold ethanol, then
eluted in water from a NAPTM10 column (Sephadex G-25 DNA
grade resin, Pharmacia) to give 3.0 A260 units of 2. On negative
ion electrospray mass spectrometry, the synthetic conjugate
gave rise to a series of negative ions whose deconvolution
predicted a molecular weight of 5592.7; structure 2 requires
5592.8.
dryness. The residue was extracted with hexane to remove excess
benzil and was then purified by flash column chromatography
using chloroform–ethanol (15 : 1) as eluant to give 2-amino-
4- (3-c◦hloropropoxy)-6,7-diphenylpteridine 9 (74 mg, 21%), mp
>278 C (decomp.) (from CHCl3/EtOH) (found: C, 64.41; H,
4.44; Cl, 8.87; N, 17.82. Calc. for C21Hl8ClN5O: C, 64.37; H, 4.63;
Cl, 9.05; N, 17.87%); kmax(EtOH)/nm 226 (log e/dm3 mol−1 cm−1
4.47), 240 (4.43), 288 (4.31) and 388 (4.14); mmax/cm−1 3460,
3260, 3080, 1630, 1590, 1530, 1240, 1170, 1150, 1090, 1005, 825,
790, 765, 725 and 700; dH (80 MHz; d6-DMSO) 2.32 (2 H, m,
CH2CH2Cl), 3.83 (2 H, t, J 6.5, CH2Cl), 4.64 (2 H, t, J 6.2,
OCH2-), 7.34 and 7.38 (12 H, 2 × s, NH2 and 6,7-diPh).
Pteridinone–oligodeoxynucleotide conjugate 3
The 5ꢀ-aminohexyl modified 17-mer reagent was prepared
(1 lmol scale) on the automated DNA synthesiser by ter-
minal addition of 5ꢀ-AminoModifier phosphoramidite (Glen
Research) to the 17-mer. It was purified using a reverse phase
cartridge (PolyPak, Transgenomics) and converted to its lithium
salt by precipitation from LiCl solution with ethanol. A so-
lution of 2-(6-succinimidocarboxypentylamino)-6,7-diphenyl-
4(3H)-pteridinone 37 (15 mg, 0.029 mmol) in anhydrous DMF
(150 ll) was added to an Eppendorf tube containing 16.0
A260 units of the aminohexyl modified 17-mer dissolved in
100 mM sodium–borate buffer, pH 8.8 (100 ll). The mixture
was vortexed and heated at 40 ◦C for 1 h. The reaction mixture
was then passed through a NAPTM10 column and eluted with
water (2 × 1 cm3). The fractions were pooled, concentrated,
and then purified by successive denaturing polyacrylamide
gel electrophoresis and electroelution steps as described for
conjugate 2 above. The recovered solution of 3 in TRIS–borate
buffer was desalted and concentrated by repeated extraction with
1-butanol prior to ethanol precipitation from sodium acetate
solution. When dissolved in water, the pale yellow pellet afforded
4.7 A260 units of product 3. Deconvolution of its negative ion
electrospray mass spectrum gave a predicted molecular weight
of 5749.2; structure 3 requires 5748.1.
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-
pteridinone 10
A suspension of 5-amino-7-(3-chloropropoxy)furazano[3,4-
d]pyrimidine 5 (200 mg, 0.87 mmol) and 10% palladised carbon
(50 mg) in deaerated water (18.5 cm3) was stirred vigorously
under hydrogen at atmospheric pressure until hydrogen uptake
ceased. The catalyst was removed by vacuum filtration under an
argon atmosphere. A solution of potassium hydroxide (123 mg,
2.19 mmol) and benzil (387 mg, 1.92 mmo1) in deaerated
methanol (18.5 cm3) was added and the resulting suspension
◦
was stirred at 40 C for 1.5 h. The suspension was neutralised
with dilute HCl and the solvent evaporated to give a yellow
residue which was extracted with methanol. Evaporation of
the methanol extract gave a solid, which on flash chromatog-
raphy using chloroform–ethanol (5 : 1) as eluant, gave 2-
amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10
as a yellow solid (216 mg, 67%), mp 264 ◦C (from EtOH/H2O)
(found: C, 66.14; H, 5.09; N, 18.21. Calc. for C21H19N5O2(0.5
H2O): C, 65.96; H, 5.27; N, 18.31%); kmax(EtOH)/nm 226 (log
e/dm3 mol−1 cm−1 4.38), 297.5 (4.36) and 382 (4.05); mmax/cm−1
3372, 3130, 1692, 1669, 1543 and 1528; dH (300 MHz; d6-DMSO)
1.83 (2 H, m, CH2CH2OH), 3.52 (2 H, m, CH20H), 4.09 (2 H,
t, J 7.0, CH2N), 4.68 (l H, t, J 5.0, OH), 7.29–7.43 (10 H, m,
6,7-diPh), 7.54 (2 H, br s, NH2).
5-Amino-7-(3-chloropropoxy)furazano[3,4-d]pyrimidine 5
5-Amino-7-(methylthio)furazano[3,4-d]pyrimidine26 4 (1.88 g,
10.3 mmol) was suspended in 3-chloropropanol (24 cm3) and
heated to 55 ◦C in a flask with a drying tube and dropping funnel
attached. A 30% w/v solution of bromine in 3-chloropropanol
was added dropwise, keeping the temperature at 55 ◦C. Each
successive addition was made when the bromine colour from the
previous addition had disappeared, and addition was continued
until the solution maintained a permanent bromine colour. The
reaction mixture was cooled to room temperature and poured
with vigorous stirring on to ethyl acetate (500 cm3) containing
sodium hydrogen carbonate (8 g). The mixture was stirred for
10 min, saturated aq sodium chloride (40 cm3) was added, and
the mixture stirred for a further 10 min. The organic layer
was dried (MgSO4) and evaporated in vacuo to give a yellow
oil. Diethyl ether (20 cm3) was added and the solution stored
at −5 ◦C overnight. 5-Amino-7-(3-chloropropoxy)furazano[3,4-
d]pyrimidine, 5 was obtained as a yellow solid (1.59 g, 69%), mp
178–80 ◦C (found (tungstic oxide): C, 36.48; H, 3.45; Cl, 15.73;
N, 30.42. Calc. for C7H8ClN5O: C, 36.61; H, 3.51; Cl, 15.44;
N, 30.50%); kmax(EtOH)/nm 214 (log e/dm3 mol−1 cm−1 4.21),
255 (3.68) and 339 (3.55); mmax/cm−1 3410, 870, 840 and 785; dH
(400 MHz; d6-DMSO) 2.32 (2 H, m, CH2CH2CH2), 3.82 (2 H,
t, J 6.5, ClCH2), 4.67 (2 H, t, J 6.5, CH2O), 7.84 (2 H, d, NH2).
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone
10, and 2-amino-4-methoxy-6,7-diphenylpteridine 14, from
5-amino-7-(3-chloropropoxy)furazano[3,4-d]pyrimidine 5
A. (By hydrogenation of 5 in water and isolation of the
intermediate resonance stabilised cation 8). A suspension
of 5-amino-7-(3-chloropropoxy)furazano[3,4-d]-pyrimidine 5
(110 mg, 0.48 mmol) and 10% palladised carbon in deaerated
water was stirred overnight under hydrogen at room temperature
and pressure. The catalyst was filtered off under nitrogen and
the filtrate evaporated to dryness at room temperature under
reduced pressure using an oil pump. The resulting solid 8
was dissolved in deaerated methanol (15 cm3) under nitrogen.
Sodium (15 mg, 0.65 mmol) and benzil (190 mg, 1.10 mmol)
were added, and the solution was refluxed under nitrogen for
3 h. The mixture was allowed to cool, neutralised with dilute
methanolic sulfuric acid, filtered, and evaporated to dryness
under reduced pressure. Flash chromatography of the residue
on silica using chloroform–ethanol (gradient from 50 : 1–7 :
1) as eluant yielded 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-
4(3H)-pteridinone 10 (64 mg, 35%), identical in properties
with the sample prepared as described above, and 2-amino-4-
methoxy-6,7-diphenylpteridine 14 (5 mg, 3%).26
2-Amino-4-(3-chloropropoxy)-6,7-diphenylpteridine 9
B. (By hydrogenation of 5 in methanol). A mixture
A suspension of 5-amino-7-(3-chloropropoxy)furazano[3,4-d]-
pyrimidine 5 (205 mg, 0.89 mmol), 10% palladised carbon
(40 mg) in tetrahydrofuran (20 cm3) was deaerated with nitrogen
and then stirred for 36 h at room temperature under an
atmosphere of hydrogen. The reaction flask was flushed with
nitrogen, benzil (500 mg, 2.38 mmol) was added, and the
mixture was then refluxed under nitrogen for 18 h. The reaction
mixture was cooled and filtered, and the filtrate evaporated to
of 5-amino-7-(3-chloropropoxy)furazano[3,4-d]pyrimidine 5
(200 mg, 0.87 mmol) and 10% palladised carbon (48 mg) in
methanol (30 cm3) was stirred at room temperature under an
atmosphere of hydrogen for 2 h. The flask was transferred to
an atmosphere of nitrogen, and sodium (30 mg, 1.3 mmol)
was added. When the sodium had dissolved, benzil (380 mg,
1.81 mmol) was added and the mixture refluxed with stirring
under nitrogen for 3 h. After cooling, the mixture was neutralised
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 9 5

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with dilute methanolic sulfuric acid, filtered, and evaporated to
dryness under a reduced pressure. Flash column chromatogra-
phy of the residue using chloroform–ethanol (50 : 1–100 : 15)
as eluant yielded 2-amino-4-methoxy-6,7-diphenylpteridine 14
(90 mg, 31%)26, and 10 (66 mg, 20%), identical with that prepared
as described above.
with stirring under nitrogen for 15 h. After cooling, the reaction
mixture was evaporated under reduced pressure and the residue
taken up in chloroform, washed with water, dried (MgSO4) and
evaporated. Flash chromatography using chloroform as eluant
separated the products from polar impurities. Chromatography
of the products on a second column, using progressively more
polar solvent mixtures as eluant (ethyl acetate–hexane, 1 : 5–1
: 1; ethyl acetate–chloroform, 1 : 1; chloroform; chloroform–
ethanol, 10 : 1) allowed the isolation of two products,
2-amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone 18
(43 mg, 39%), identical with the product obtained from 10 under
different conditions as described below, and 3-(3-chloropropyl)-
2-Amino-3-(3-hydroxypropyl)-6,7-dimethyl-4(3H)-pteridin-
one 12
A
mixture of 5-amino-7-(3-chloropropoxy)furazano[3,4-
d]pyrimidine 5 (100 mg, 0.43 mmol), 10% palladised carbon
(36 mg) and deaerated water (20 cm3) was stirred overnight
at room temperature under an atmosphere of hydrogen. The
catalyst was filtered off under nitrogen by means of a filter stick
and the filtrate heated to 70 ◦C under nitrogen. Biacetyl (0.25 g,
2.9◦mmol) was added and the mixture stirred and heated to
70 C for a further 2 h. Tetrahydrofuran (20 cm3) was added at
room temperature with stirring, and examination after 1 h by
TLC (chloroform–ethanol, 15 : 2) showed complete conversion
to a polar fluorescent material (Rf 0.2). The solution was
evaporated to dryness and flash chromatography of the residue
using chloroform–ethanol (50 : 1–100 : 15) as eluant yielded
2-amino-3-(3-hydroxypropyl)-6,7-dimethyl-4(3H)-pteridinone
12 (80 mg, 74%), mp >220 ◦C (decomp.) (from EtOH) (found:
C, 53.07; H, 5.96; N, 27.86. Calc. for C11H15N5O2: C, 53.00;
H, 6.07; N, 28.10%); kmax (EtOH 95%)/nm 225 infl (log
e/dm3 mol−1 cm−1 3.97), 243 (4.07), 279 (4.07) and 354 (3.74);
mmax/cm−1 (nujol) 3560 (NH2), 3380 (OH), 1720 and 1680
(C=O and C=N); dH (80 MHz; d6-DMSO) 1.78 (2 H, m,
CH2CH2OH), 2.504 and 2.496 (6 H, 2 s, 2 × CH3), 3.49 (2 H,
m, CH2OH), 4.04 (2 H, t, J 7.0, NCH2), 4.72 (1 H, t, J 5.0,
OH) and 7.31 (2 H, s, NH2).
6,7-diphenyl-2-triphenylphosphazino-4(3H)-pteridinone
15
(85 mg, 46%), mp 191–93 ◦C (from ethyl acetate–hexane)
(found (using tungstic oxide): C, 71.76; H, 4.92; Cl, 5.78; N,
10.58. Calc. for C39H31C1N5OP: C, 71.83; H, 4.79; Cl, 5.44;
N, 10.74%); mmax/cm−1 3070, 1700 (C=O), 1530, 1515, 1190,
1115, 990 and 725; kmax (EtOH)/nm 261 (log e/dm3 mol−1 cm−1
4.29), 314 (4.51) and 390 (4.07); kmax (1,4-dioxane)/nm 229
(log e/dm3 mol−1 cm−1 4.60), 320 (4.44) and 381 (4.09); dH
(300 MHz; CDCl3) 2.38–2.48 (2 H, m, CH2CH2Cl), 3.77 (2 H,
t, J 6.4, CH2Cl), 4.73 (2 H, t, J 7.2, NCH2) and 7.18–8.00 (25
H, m, Ph3P and 6,7-di-Ph); dC (75.5 MHz, CDCl3) 31.06, 41.94,
43.33, 126.98, 127.08, 127.88, 127.98, 128.34, 128.60, 128.76,
128.86, 129.91, 130.00, 132.52, 133.53, 133.66, 133.90, 147.61,
153.32, 157.30 and 162.81; dP (121.5 MHz, CDCl3) 20.4 (s); m/z
(EI) 651.1958 (M+, 27%, C39H31C1N5OP requires 651.1955),
574.1778 (0.4, M+ − C3H6Cl), 276.0919 (3, C18H15NP), 262.0914
(100, C18H15P), 185.0533 (21, C12H10P), 108.0133 (32, C6H5P)
and 77.0398 (19, C6H5).
6,7-Diphenyl-2-phthalimido-3-(3-phthalimidopropyl)-4(3H)-
pteridinone 16
3-(3-Chloropropyl)-6,7-diphenyl-2-phthalimido-4(3H)-
2-Amino-3-(3-hydroxypropyl)-7-methyl-4(3H)-pteridinone 13
pteridinone 19 (50 mg, 0.96 mmol) and potassium phthalimide
(34 mg, 0.184 mmol) in hexamethylphosphoramide were heated
under nitrogen for 40 min. The mixture was cooled and acidified
with dilute hydrochloric acid (0.5 cm3), and was then added to
ethyl acetate, washed with water, dried (MgSO4), and evaporated
under reduced pressure. Chromatography on a short column
using dichloromethane–ethanol (150 : 1) as eluant removed
some highly polar impurities, and crystallisation of the crude
product thus obtained from dichloromethane/hexane/ethyl
acetate afforded pure 6,7-diphenyl-2-phthalimido-3-(3-
phthalimidopropyl)-4(3H)-pteridinone 16 as white crystals
(15 mg, 22%), mp 286–290 ◦C (found: C, 68.16; H, 4.38; N,
11.65. Calc. for C37H24N6O5/EtOAc: C, 68.33; H, 4.47; N,
11.66%); kmax (1,4-dioxane)/nm 218 (log e/dm3 mol−1 cm−1
4.64), 265 (4.36) and 350 (4.17); mmax/cm−1 3059, 1791, 1766,
1733, 1710, 1592, 1531, 1407, 1246, 1186, 1102, 1024, 959, 881,
720 and 703; dH (300 MHz; CDCl3) 1.26 (3 H, t, J 7.1, EtOAc),
2.04 (3H, s, EtOAc), 2.22 (2 H, m, CH2CH2CH2), 3.76 (2 H, t,
J 6.2, CH2-phth), 4.12 (2 H, q, J 7.2, EtOAc), 4.24 (2 H, t, J
8.0, CH2-pteridine), 7.26–7.60 (10 H, m, 6,7-diPh), 7.66 (4 H, s,
phth) and 7.78–7.88 (4 H, m, phth); dC (75.5 MHz, CDCl3) 14.18
(EtOAc), 21.03 (EtOAc), 27.87, 35.32, 43.58, 60.37 (EtOAc),
123.26, 124.58, 128.23, 128.38, 129.47, 129.74, 130.02, 130.18,
131.42, 131.73, 133.91, 133.25, 137.12, 137.53, 144.15, 150.53,
154.69, 158.74, 160.68, 165.36, 168.00 and 171.13 (EtOAc); m/z
(EI) 632.1796 (M+, 65%), 486.1575 (16, M+ − N(CO)2C6H4),
A
mixture of 5-amino-7-(3-chloropropoxy)furazano[3,4-
d]pyrimidine 5 (100 mg, 0.43 mmol), 10% palladised carbon
(36 mg) and deaerated water (25 cm3) was stirred overnight
at room temperature under an atmosphere of hydrogen. The
catalyst was filtered off under a nitrogen atmosphere by means
of a filter stick. The filtrate was cooled in ice and pyruvaldehyde
(27% aq solution, 1 cm3, 3.8 mmol) added under nitrogen. The
mixture was stirred for 2 h at 0–5 ◦C, when examination by
TLC (chloroform–ethanol, 15 : 2) showed a fluorescent spot of
very low Rf. The mixture was concentrated in vacuo with gentle
warming to about one fifth of its volume, tetrahydrofuran
(25 cm3) added under nitrogen, and the mixture stirred at
room temperature for a further 1.5 h. Examination by TLC
(chloroform–ethanol, 15 : 2) showed a new polar product
(Rf 0.2) and evaporation of the mixture to dryness under
reduced pressure followed by flash chromatography using
chloroform–ethanol (20 : 1–2 : 1) as eluant yielded 13 (70 mg,
69%), mp >220 ◦C (decomp.) (from ethanol) (found: C, 50.69;
H, 5.30; N, 29.61. Calc. for C10H13N5O2: C, 51.06; H, 5.57; N,
29.77%); kmax (EtOH)/nm 243 (log e/dm3 mol−1 cm−1 4.17), 280
(4.08) and 353 (3.82); mmax/cm−1 3430, 3300, 1675, 1640 and
1540; dH (300 MHz; d6-DMSO) 1.78 (2 H, m, CH2CH2OH),
2.52 (3 H, s, CH3), 3.48 (2 H, m, CH2OH), 4.02 (2 H, t, J 7.2,
NCH2), 4.67 (l H, t, J 5.0, OH), 7.44 (2 H, br s, NH2) and 8.27
(l H, s, 6H); dC (75.5 MHz, d6-DMSO) + DEPT 21.87 (CH3),
30.03 (CH2), 38.66 (CH2), 58.03 (CH2), 125.29 (C), 139.27
(CH), 154.08 (C), 154.87 (C), 159.32 (C) and 160.67 (C).
472.1459 (18, M+ − CH2N(CO)2C6H4), 444.1176 (14, M+
−
(CH2)3N(CO)2C6H4) and 77.0394 (12, C6H5).
3-(3-Chloropropyl)-6,7-diphenyl-2-triphenylphosphazino-4(3H)-
pteridinone 15 and 2-amino-3-(3-chloropropyl)-6,7-diphenyl-
4(3H)-pteridinone 18
2-(3-Hydroxypropylamino)-6,7-diphenyl-4(3H)-pteridinone 17
A
solution of 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-
4(3H)-pteridinone 10 (940 mg, 2.46 mmol) and potassium hy-
droxide (33 g, 0.59 mol) in methanol–water (1 : 1) (300 cm3) was
refluxed for 3 h and allowed to cool. The mixture was neutralised
with dilute aqueous sulfuric acid and stored at 5 ◦C overnight.
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone
10 (107 mg, 0.28 mmol), triphenylphosphine (481 mg,
1.84 mmol) and tetrachloromethane (27 cm3) were refluxed
3 5 9 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1

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The resulting solid material was filtered off and extracted
with methanol, the extracts were combined and concentrated
under reduced pressure, when 2-(3-hydroxypropylamino)-6,7-
diphenyl-4(3H)-pteridinone 17 crystallised out (600 mg, 65%),
mp 275–76 ◦C (from ethanol) (found: C, 67.22: H, 4.94; N,
18.48. Calc. for C21H19N5O2: C, 67.55; H, 5.13; N, 18.75%);
kmax (EtOH)/nm 224 (log e/dm3 mol−1 cm−1 4.36), 250 (4.25),
299 (4.38) and 381 (4.05); kmax (aq KOH 0.1M)/nm 219
(log e/dm3 mol−1 cm−1 4.32) 279 (4.36) and 389 (4.09); dH
(300 MHz; d6-DMSO) 1.74 (2 H, m, –CH2CH2OH), 3.52 (4
H, m, CH2CH2CH2OH), 6.33 (2 H, 2 × br s, D2O exchangeable,
–CH2NH and –OH), 7.34 and 7.38 (10 H, 2 × s, 6,7-diPh) and
11.36 (1 H, br s, D2O exchangeable, CONH).
solution. After 30 min, water (10 cm3) was added to the reaction
mixture, followed by evaporation of most of the ethanol under
reduced pressure. The aqueous residue was extracted with
chloroform and the extract washed with water, dried (MgSO4),
and evaporated under reduced pressure, to give 2,3-diphenyl-
6,7,8,9-tetrahydropyrimido[2,1-b]pteridine-11-one 20 (35 mg,
◦
78%), mp >310 C (decomp.) (from chloroform–ethyl acetate)
(found: C, 70.53; H, 4.81; N, 19.40. Calc. for C21H17N5O:
C, 70.97; H, 4.82; N, 19.71%); kmax (EtOH)/nm 226 (log
e/dm3 mol−1 cm−1 4.35), 250 (4.27), 301 (4.38) and 390 (4.01);
mmax/cm−1 1687, 1639, 1546, 1527, 1316, 1287, 1245, 1188,
1069, 972, 789, 778 and 699; dH (300 MHz; CDCl3) 2.12 (2 H,
m, CH2CH2CH2), 3.64 (2 H, t, J 5.4, CH2), 4.18 (2 H, t, J
5.9, CH2), 7.23–7.51 (10 H, m, 2,3-diPh) and 9.01 (1 H, br s,
NH); dC (75.5 MHz, CDCl3) 2.05, 39.39, 40.53, 125.82, 128.00,
128.09, 128.29, 129.94, 138.42, 138.56, 148.44, 152.45, 153.79,
158.06 and 161.02.
B. 2-(3-Hydroxypropylamino)-6,7-diphenyl-4(3H)-pteridin-
one 17 (210 mg, 0.56 mmol), triphenylphosphine (1.1 g,
4.2 mmol) and tetrachloromethane (80 cm3) were refluxed
together with stirring under nitrogen for 12 h. The reaction
mixture was cooled and evaporated under reduced pressure.
Flash chromatography, eluting with chloroform followed by
chloroform–ethanol (50 : 1) afforded 2,3-diphenyl-6,7,8,9-
tetrahydropyrimido[2,1-b]pteridine-11-one 20 (39 mg, 20%),
identical in properties with that prepared as described above.
2-Amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone 18
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone
10 (214 mg, 0.56 mmol) and triphenylphosphine (1.74 g,
6.63 mmol) were dissolved in dry tetrahydrofuran (60 cm3)
with heating under nitrogen. Tetrachloromethane (60 cm3)
was added and the solution heated to reflux for 30 min.
The reaction mixture was cooled and then evaporated under
reduced pressure. Flash chromatography of the residue, using
successively as eluant ethyl acetate, ethyl acetate–ethanol (15 :
1), chloroform, and finally chloroform–ethanol (100 : 1), gave
2-amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone 18
(144 mg, 66%), mp >300 ◦C (decomp) (found: C, 64.16; H, 4.87,
Cl, 8.78; N, 17.48. Calc. for C21H18ClN5O: C, 64.37; H, 4.63; Cl,
9.05; N, 17.87%); kmax (EtOH)/nm 225 (log e/dm3 mol−1 cm−1
4.40) 245 infl (4.28), 297 (4.37) and 382 (4.05); mmax/cm−1
3490, 3300, 3050, 1710 (C=O), 1655, 1550, 1195 and 695; dH
(300 MHz; d6-DMSO) 2.35 (2 H, m, CH2CH2Cl), 3.75 (2 H, t,
J 5.7, CH2Cl), 4.29 (2 H, t, J 7.3, NCH2), 5.64 (2 H, br s, NH2)
and 7.29–7.57 (10 H, m, 6,7-diPh).
2-Acetamido-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-
pteridinone 21
2-Amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone
18 (100 mg, 0.26 mmol) was dissolved in dry pyridine (8 cm3)
in a closed flask. Acetic anhydride (27 ll, 29 mg, 0.28 mmol)
was added and the reaction mixture was stirred in the closed
flask at room temperature for 70 h. The pyridine was then
evaporated under reduced pressure. Flash chromatography of
the residue with chloroform–ethanol (200 : 1) as eluant yielded a
product that was rechromatographed on a second column using
ethyl acetate–hexane (2 : 5) as eluant. Fractions containing
the first eluted product were combined and evaporated, to
3-(3-Chloropropyl)-6,7-diphenyl-2-phthalimido-4(3H)-
pteridinone 19
2-Amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone
18 (100 mg, 0.26 mmol), phthalic anhydride (1.6 g, 10.8 mmol)
and dry pyridine (5 cm3) were heated to 50 ◦C with stirring
under nitrogen for 5 h. The reaction mixture was cooled,
added to ethyl acetate (150 cm3) and washed with water (6 ×
50 cm3). The organic layer was dried (MgSO4) filtered, and
evaporated under reduced pressure. Flash chromatography
of the residue, using as eluant dichloromethane followed by
dichloromethane–ethanol (200 : 1), yielded 3-(3-chloropropyl)-
6,7-diphenyl-2-phthalimido-4(3H)-pteridinone 19 (92 mg,
69%), mp 269–70 ◦C (from dichloromethane–hexane) (found:
C, 67.08; H, 3.88; Cl, 6.87; N, 13.28. Calc. for C29H20C1N503:
C, 66.73; H, 3.86; Cl, 6.79; N, 13.42%); kmax (1,4-dioxane)/nm
265 (log e/dm3 mol−1 cm−1 4.35), 350 (4.18); mmax/cm−1 1795,
1734, 1707, 1594, 1538, 1406, 1365, 1322, 1244, 1225, 1107,
955, 883, 857, 793, 722 and 697; dH (300 MHz; CDCl3) 2.28 (2
H, m, CH2CH2Cl), 3.59 (2 H, t, J 5.8, CH2Cl), 4.36 (2 H, t,
J 7.2, NCH2), 7.22–7.62 (10 H, m, 6,7-di-Ph), 7.88 (2 H, dd,
J 5.6 and 3.2, phth) and 8.02 (2 H, dd, J 5.6 and 3.2, phth);
dC (75.5 MHz, CDCl3) + DEPT 30.92 (CH2), 42.04 (CH2),
43.08 (CH2), 124.7 (CH), 128.28 (CH), 128.42 (CH), 129.54
(CH), 129.70 (C), 130.02 (CH), 130.20 (CH), 131.62 (C), 135.40
(CH), 137.11 (C), 137.55 (C), 144.24 (C), 150.69 (C), 154.74
(C), 158.86 (C), 160.99 (C) and 165.61 (C).
◦
afford 21 (36 mg, 33%), mp 178 C (decomp.) (from acetone–
hexane) (found: C, 64.07; H, 4.65; Cl, 8.22; N, 16.32. Calc.
for C23H20ClN5O2: C, 63.67; H, 4.65, Cl, 8.17; N, 16.14%);
kmax (1,4-dioxane)/nm 234 (log e/dm3 mol−1 cm−1 4.33), 273
(4.37) and 368 (4.23); mmax/cm−1 3413 (NH), 3061, 1716 (C=O),
1593, 1546, 1280, 1252, 1196, 1121, 1010, 978, 774 and 695; dH
(300 MHz; CDC13) 2.28 (2 H, m, CH2CH2Cl), 2.33 (3 H, s,
COCH3), 3.67 (2 H, t, J 6.6, CH2Cl), 4.50 (2 H, t, J 7.0, NCH2)
and 7.27–7.53 (10 H, m, 6,7-diPh).
3-(3-Chloropropyl)-2-(2-ethoxycarbonylbenzamido)-6,7-
diphenyl-4(3H)-pteridinone 22
3-(3-Chloropropyl)-6,7-diphenyl-2-phthalimido-4(3H)-pteri-
dinone 19 (55 mg, 0.11 mmol) was dissolved in dichloromethane
(2.0 cm3). Ethanol (10 cm3) followed by dilute aqueous sodium
hydroxide (0.2 cm3) were added and the mixture was stirred for
2 min. Purification was effected by preliminary chromatography
of the crude reaction mixture on a short silica column, eluting
with dichloromethane–ethanol (10 : 1), followed by flash
chromatography eluting with dichloromethane–ethanol (100 :
1), to give 3-(3-chloropropyl)-2-(2-ethoxycarbonylbenzamido)-
6,7-diphenyl-4(3H)-pteridinone 22 (54 mg, 91%), mp 180–81 ◦C
(from acetone–hexane) (found: C, 65.65; H, 4.69; Cl, 6.12; N,
12.35. Calc. for C31H26ClN5O4: C, 65.55; H, 4.61; Cl, 6.24; N,
12.33%); kmax (1,4-dioxane)/nm 306 (log e/dm3 mol−1 cm−1
4.46) and 370 (4.38); mmax/cm−1 1727 (C=O), 1712 (C=O), 1581,
1548, 1357, 1313, 1287, 1259, 1193, 1120, 1035, 746 and 699;
dH (300 MHz; CDCl3) 1.37 (3 H, t, J 7.2, CH3), 2.32 (2 H, m,
CH2CH2Cl), 3.69 (2 H, t, J 6.5, CH2Cl), 4.41 (2 H, q, J 7.2,
CH2CH3), 4.58 (2 H, t, J 7.1, NCH2), 7.29–7.63 (13 H, m,
2,3-Diphenyl-6,7,8,9-tetrahydropyrimido[2,1-b]pteridine-
11-one 20
A. 2-Amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridin-
one 18 (50 mg, 0.13 mmol) was dissolved in ethanol (20 cm3)
with heating and the solution was then cooled to room
temperature. Sodium hydroxide (0.1 g, 2.6 mmol) was dissolved
in water and this solution added to the ethanolic pteridine
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 9 7

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6,7-diPh and Ar) and 8.13–8.17 (1 H, m, Ar); dC (75.5 MHz,
CDCl3) + DEPT 14.09 (CH3), 30.38 (CH2), 41.01 (CH2), 42.36
(CH2), 61.42 (CH2), 125.42 (C), 128.05 (CH), 128.32 (CH),
128.35 (CH), 129.19 (CH), 129.70 (CH), 129.75 (CH), 129.92
(CH), 130.14 (CH), 130.32 (CH), 131.06 (CH), 133.68 (C),
136.52 (C), 136.95 (C), 137.09 (C), 143.54 (C), 152.02 (C),
152.93 (C), 157.85 (C), 159.52 (C), 169.33 (C) and 179.70 (C).
neutralised with dilute aqueous hydrochloric acid. Most of the
methanol was evaporated under reduced pressure. The aqueous
residue was extracted with chloroform, and the extract dried
(MgSO4), filtered, and concentrated under reduced pressure to
a volume of about 30 cm3 to give a solution of crude 23. To this
solution were added acetic anhydride (0.5 cm3) and pyridine
(0.5 cm3), and the mixture was stirred under nitrogen at room
temperature for 1 h. It was then washed with water, and the
organic layer was dried (MgSO4) and evaporated under reduced
pressure. Flash chromatography of the residue on silica, using
dichloromethane–ethanol (100 : 1) as eluant, gave 26 (29 mg,
32%), mp 215–19 ◦C (from dichloromethane–hexane) (found:
C, 63.45; H, 5.12; N, 15.76. Calc. for C23H21N5O3 0.25CH2Cl2:
C, 63.95; H, 4.96; N, 16.04%); kmax (MeOH)/nm 226 (log
e/dm3 mol−1 cm−1 4.46), 269 (4.20) and 363 (4.19); mmax/cm−1
3319, 3080, 1734 (C=O), 1661 (C=O), 1560, 1243, 752, 720
and 697; dH (300 MHz; CDCl3) 1.96 (2 H, quintet, J 6.1,
CH2CH2CH2), 2.02 (3 H, s, COCH3), 3.27 (2 H, q, J 6.1,
CH2NH), 4.21 (2 H, t, J 6.2, CH2CH2CH2NH), 5.29 (0.5 H, s,
0.25 × CH2Cl2), 6.41(l H, t, J 5.9, CH2 NH), 7.25–7.47 (10 H,
m, 6,7-diPh) and 8.99 (l H, br s, pteridine-NH); dC (75.5 MHz;
CDCl3) + DEPT 23.4 (CH3), 27.7 (CH2), 36.0 (CH2), 39.0
(CH2), 124.1 (C), 128.4 (CH), 129.1 (CH), 129.8 (CH), 129.9
(CH), 130.3 (CH), 136.8 (C), 137.3 (C), 144.9 (C), 149.8 (C),
151.0 (C), 157.2 (C), 161.1 (C) and 170.4 (C).
6-Acetyl-2,3-diphenyl-6,7,8,9-tetrahydropyrimido[2,1-b]pteri-
dine-11-one 24
A. 2,3-Diphenyl-6,7,8,9-tetrahydropyrimido[2,1-b]pteridin-11-
one 20 (63 mg, 0.18 mmol) was added to dry pyridine (10 cm3)
followed by acetic anhydride (1.0 cm3). The mixture was heated
with stirring to 45 ◦C until the pteridine was fully dissolved
and was then stirred for 12 h at room temperature, after which
the pyridine was evaporated under a reduced pressure. Flash
chromatography of the residue, using chloroform as eluant,
followed by further chromatography on a second column eluting
with hexane–acetone (4 : 1), afforded 6-acetyl-2,3-diphenyl-
6,7,8,9-tetrahydropyrimido[2,1-b]pteridine-11-one 24 (29 mg,
41%), mp >134 ◦C (decomp.) (found: C, 69.27; H, 4.59; N,
17.35. Calc. for C23H19N5O2: C, 69.51; H, 4.82; N, 17.62%);
kmax (EtOH)/nm 255 (log e/dm3 mol−1 cm−1 4.27), 304 (4.29)
and 366 (4.13); mmax/cm−1 3060, 1691 (C=O), 1583, 1531, 1412,
1294, 1261, 1211, 1153, 1036, 973, 774 and 695; dH (300 MHz;
CD3COCD3) 2.25–2.34 (2 H, m, CH2CH2CH2), 2.65 (3 H, s,
COCH3), 3.92 (2 H, t, J 6.7, CH2), 4.21 (2 H, t, J 5.8, CH2)
and 7.32–7.65 (10 H, m, 6,7-diPh); dC (75.5 MHz, CD3COCD3)
32.24, 36.09, 50.72, 53.25, 138.60, 138.65, 139.18, 139.88, 140.36,
140.46, 148.96, 149.1, 161.4, 161.6, 162.2, 168.3, 170.1 and 182.4.
2,3-Diphenyl-5,8,9,10-tetrahydropyrimido[1,2-c]pteridine-6-
one 27
2,3-Diphenyl-6,7,8,9-tetrahydropyrimido[2,1-b]pteridin-11-one
20 (116 mg, 0.33 mmol), methanol (20 cm3), water (10 cm3)
and potassium hydroxide (4.0 g) were heated together under
reflux for 5.5 h. The mixture was cooled and then neutralised
with dilute aqueous hydrochloric acid. Most of the methanol
was evaporated under a reduced pressure and the aqueous
residue was extracted with chloroform. The extract was dried
(MgSO4) and evaporated under a reduced pressure, to give a
mixture of 23 and 27. Flash chromatography, eluting first with
dichloromethane–ethanol (15 : 1, 15 : 2 and 5 : 1) and then with
methanol, yielded 27, together with the more polar 23. The latter
on refluxing in methanol solution for a further 3 h, followed by
workup and chromatography gave more 27. The combined yield
of 2,3-diphenyl-5,8,9,10-tetrahydropyrimido[1,2-c]pteridine-6-
one 27 was 55 mg (47%), kmax (EtOH)/nm 297, 383 and 406;
kmax (0.1 M aq KOH)/nm 301 and 399, kmax (0.1 M aq HCl)/nm
278 and 379; mmax/cm−1 3120, 1700 (C=O), 1640, 1560, 1280,
1190, 1100, 1035, 980, 810, 770, 730 and 700; dH (300 MHz;
CDCl3) 2.03 (2 H, m, CH2CH2CH2), 3.82 (2 H, t, J 5.6, CH2N),
4.00 (2 H, t, J 5.9, CH2N) and 7.2–7.4 (10 H, m, 6,7-diPh); dC
(75.5 MHz, CDCl3) + DEPT 20.03 (CH2), 41.37 (CH2), 44.94
(CH2), 126.35 (C), 128.21 (CH), 128.27 (CH), 128.54 (CH),
129.51 (CH), 129.81 (CH), 129.85 (CH), 137.37 (C), 137.94 (C),
144.21 (C), 144.91 (C), 149.28 (C), 149.40 (C) and 154.48 (C);
m/z (EI) 355 (100%, M+), 299 (2, M+ − C3H6N) and 77 (7,
C6H5).
B.
2-Acetamido-3-(3-chloropropyl)-6,7-diphenyl-4-(3H)-
pteridinone 21 (16 mg, 0.037 mmol) was dissolved in tetrahydro-
furan (10 cm3), and dilute aqueous sodium hydroxide (0.25 cm3)
was then added to the solution. The mixture was stirred at
room temperature for 2.5 h, and was then evaporated under a
reduced pressure. Flash chromatography of the residue, using
dichloromethane as eluant followed by a dichloromethane–
ethanol gradient (150 : 1–75 : 1) afforded 6-acetyl-2,3-diphenyl-
6,7,8,9-tetrahydropyrimido[2,1-b]pteridine-11-one 24 (7 mg,
49%), identical in properties to the sample prepared as described
above.
3-(3-Chloropropyl)-2-diacetylamino-6,7-diphenyl-4(3H)-
pteridinone 25
2-Amino-3-(3-chloropropyl)-6,7-diphenyl-4(3H)-pteridinone 18
(99 mg, 0.25 mmol), dry pyridine (7 cm3) and acetic anhydride
(5 cm3, 5.4 g, 0.053 mol) were stirred together at room
temperature for 5 h. The mixture was added to ethyl acetate
(100 cm3) and the solution washed with water. The organic
layer was dried (MgSO4) and evaporated under reduced pressure.
Flash chromatography, eluting first with chloroform and then
with chloroform–ethanol (15 : 1), gave 3-(3-chloropropyl)-2-
diacetylamino-6,7-diphenyl-4(3H)-pteridinone 25 (86 mg, 72%),
mp >164 ◦C (decomp.) (from acetone–hexane) (found: C, 63.04;
H, 4.60; Cl, 8.00; N, 14.72. Calc. for C25H22ClN5O3: C, 63.09;
H, 4.66; Cl, 7.45; N, 14.72%); kmax (l,4-dioxane)/nm 236 (log
e/dm3 mol−1 cm−1 4.37), 265 (4.33) and 351 (4.17); mmax/cm−1
3060, 1740 (C=O), 1705 (C=O), 1595, 1530, 1400, 1240, 1215,
1045, 980, 955, 800, 790 and 700; dH (300 MHz; CDCl3) 2.29 (2
H, m, ClCH2CH2), 2.47 (6 H, s, 2 × COCH3), 3.67 (2 H, t, J
5.9, ClCH2), 4.21 (2 H, t, J 7.8, CH2N) and 7.25–7.64 (10 H, m,
6,7-diPh).
6,7-Diphenyl-2-triphenylphosphazino-4(3H)-pteridinone 29
2-Amino-4-methoxy-6,7-diphenylpteridine
14
(50
mg,
0.15 mmol) and triphenylphosphine (800 mg, 3.05 mmol)
were dissolved in a mixture of tetrachloromethane (20 cm3) and
tetrahydrofuran (5 cm3) and the solution refluxed for 1.2 h,
after which time complete reaction was indicated by TLC. (No
reaction occurred in the absence of tetrahydrofuran, even after
6 h refluxing). The reaction mixture was cooled, evaporated
under a reduced pressure, and purified by flash chromatography,
using ethyl acetate–hexane (1 : 1–3 : 1) as eluant, giving 6,7-
diphenyl-2-triphen◦ylphosphazino-4(3H)-pteridinone 29 (52 mg,
60%), mp 189–91 C (from acetone–hexane) (found: C, 74.31;
H, 4.63; N, 11.64. Calc. for C36H26N5OP(0.5 H2O): C, 73.96; H,
4.65; N, 11.98%); kmax (EtOH)/nm 260 (log e/dm3 mol−1cm−1
3-(3-Acetamidopropyl)-6,7-diphenyl-2,4(1H,3H)-
pteridinedione 26
2,3-Diphenyl-6,7,8,9-tetrahydropyrimido[2,1-b]pteridin-11-one
20 (7 3 mg, 0 21 mmol), methanol (15 cm3), water (8.0 cm3)
and potassium hydroxide (3.0 g) were placed in a flask and
refluxed for 14 h. The reaction mixture was cooled and then
3 5 9 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1

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4.31), 314 (4.54) and 385 (412); mmax/cm−1 3050, 1690 (C=O),
1580, 1560, 1525, 1175, 1115, 995 and 720; dH (300 MHz;
CDCl3) 7.17–7.94 (25 H, m, Ph3P and 6,7-diPh) and 9.18 (l H,
br s, NH); dP (121.5 MHz; CDCl3; H3PO4) 19.28.
(25 : 1), giving (3-(2-N,N-dimethylaminomethyleneamino-
3,4-dihydro-4-oxo-6,7-diphenyl-3-pteridinyl)-propoxy)(2-cyano-
ethoxy)(N,N-diisopropylamino)phosphine 31 (17.2 mg, 46.6%)
as a yellow solid. dH (400.13 MHz; d6-acetone) 1.18 (12 H, m,
2 × NCH(CH3)2), 2.12 (2 H, m, CH2CH2N), 2.76 (2 H, t, J
6.1, CH2CN), 3.29 (3 H, s, NCH3), 3.40 (3 H, s, NCH3), 3.65
(2 H, m, 2 × CH(CH3)2), 3.85 (4 H, m, 2 × POCH2), 4.48 (2 H,
t, J 6.9, NCH2), 7.34–7.54 (10 H, m, 6,7-diPh, 8.94 (1 H, s,
N=CHN); dP (161 MHz, d6-acetone) 148.4.
2-(N,N-Dimethylaminomethyleneamino)-3-(3-hydroxypropyl)-
6,7-diphenyl-4(3H)-pteridinone 30
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone
10 (102 mg, 0.278 mmol) was suspended in dry tetrahydro-
furan (36 cm3) under a nitrogen atmosphere. N,N-Dimethyl-
formamide dimethylacetal (39 ll, 0.295 mmol) was added via
a syringe and the reaction was stirred for 16 h. Evaporation
of solvent gave a yellow oil which was purified by flash chro-
matography, eluting with dichloromethane–ethanol (150 :
3–150 : 4) to give 2-(N,N-dimethylaminomethyleneamino)-3-(3-
hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 30 as a yellow
solid (73 mg, 62%); dH (400.13 MHz; d6-DMSO) 2.07 (2 H, m,
CH2CH2OH), 3.22 (3 H, s, NCH3), 3.27 (3 H, s, NCH3), 3.79
(1H, s, OH), 3.59 (2 H, t, J 6.0, CH2OH), 4.55 (2 H, t, J 7.0,
NCH2), 7.26–7.57 (10 H, m, 6,7-diPh), 9.03 (1 H, s, NCHN); dC
(100.6 MHz, d6-DMSO) + DEPT 159.5 (NCHN), 30.9, 39.9,
58.24 (3 × CH2), 35.6 and 41.68 (2 × NCH3).
(3-(2-Amino-3,4-dihydro-4-oxo-6,7-diphenyl-3-pteridinyl)-
propoxy)diisopropylaminomethoxyphosphine oxide 32
2-Amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone
10 (25 mg, 0.065 mmol) was dissolved in dry tetrahydrofuran
(3 cm3) under nitrogen, and diisopropylethylamine (100 ll,
74 mg, 0.057 mmol) was added to the solution. Chloro(diiso-
propylamino)methoxyphosphine (20 ll, 20 mg, 0.10 mmol) was
added to the mixture in several portions over a period of 7 min.
The reaction mixture was stirred at room temperature for 30 min,
when more chloro(diisopropylamino)methoxyphosphine (8 ll,
8 mg, 0.04 mmol) was added. After stirring for a further 30 min,
the mixture was added to ethyl acetate (30 cm3). The solution
was washed with saturated aqueous potassium hydrogen
carbonate, and the organic layer dried (NaSO4) and evaporated
under reduced pressure. Flash chromatography of the residue,
eluting with a gradient of chloroform–ethanol (150 : 2–150 : 3),
(3-(2-N,N-Dimethylaminomethyleneamino-3,4-dihydro-4-oxo-
6,7diphenyl-3-pteridinyl)propoxy) (2-cyanoethoxy)
(N,N-diisopropylamino)phosphoramidite 31
A. 2-(N,N-Dimethylaminomethyleneamino)-3-(3-hydroxypro-
pyl)-6,7-diphenyl-4(3H)-pteridinone 30 (30 mg, 0.07 mmol)
was dried by performing three coevaporations with dry
dichloromethane (1 cm3). 1(H)-tetrazole (2.5 mg, 0.036 mmol)
dissolved in dry acetonitrile (1 cm3) was added and the solvent
was removed by vacuum pump. Dry dichloromethane (1 cm3)
was added and the solution was stirred for 30 min under an
atmosphere of nitrogen. 2-Cyanoethyl-N,N,Nꢀ,Nꢀ-tetraisopro-
pylphosphorodiamidite (46.7 ll, 0.14 mmol) was added by
micropipette and the reaction was stirred in an icebath in the
dark for 30 min. TLC (alumina plates; dichloromethane–diiso-
propylethylamine, 25 : 1) showed the absence of starting
material. The solvent was removed by vacuum pump with
the flask wrapped in tin foil. Ethyl acetate (5 cm3) and
dichloromethane (5 cm3) were added, followed by aqueous
sodium hydrogen carbonate solution 5%, 10 cm3), when the
organic layer was extracted and dried (Na2SO4). Removal of
solvent in vacuo gave (3-(2-N,N-dimethylamino methylene-
amino-3,4-dihydro-4-oxo-6,7-diphenyl-3-pteridinyl)-propoxy)
(2-cyanoethoxy)(N,N-diisopropylamino)phosphoramid -ite 31
as a yellow solid, which was stored at −5 ◦C in the dark. dH
(400 MHz; CDCl3) 1.18 (12 H, m, 2 × CH(CH3)2), 2.06 (2 H, m,
CH2CH2CH2), 2.75 (2 H, t, J 6.1, CH2CN), 3.28 (3 H, s,
NCH3), 3.39 (3 H, s, NCH3), 3.55–3.70 (4 H, m, 2 × POCH2),
3.75–3.9 (2 H, m, 2 × CH(CH3)2), 4.68 (2 H, t, J 6.9, NCH2),
7.3–7.7 (10 H, m, 6.7-diPh), 8.93 (1 H, s, N=CHN); dP
(161.9 MHz, CDCl3), 148.4.
B. 2-(N,N-Dimethylaminomethyleneamino)-3-(3-hydroxy-
propyl)-6,7-diphenyl-4(3H)-pteridinone 30 (25 mg, 0.058 mmol)
was dissolved in dry dichloromethane (2 cm3) in a two necked
flask fitted with a septum and a gas tap, and under an
atmosphere of nitrogen. Diisopropylethylamine (225 ll,
1.25 mmol) was added to the flask, followed by 2-cyanoethyl
N,N-diisopropyl-chlorophosphoramidite (37.5 ll, 0.167 mmol)
via a micropipette. The reaction was stirred at room temperature
in the dark for 30 min and the solvent was removed by vacuum
pump. Ethyl acetate (5 cm3) and dichloromethane (5 cm3)
were added, followed by aqueous sodium hydrogen carbonate
(5%, 10 cm3). The organic layer was extracted and dried
(Na2SO4), and the solvent then removed by vacuum pump.
The crude product thus obtained was stored at −5 ◦C in
the dark. The product was purified on a column of neutral
alumina, eluting with dichloromethane–diisopropylethylamine,
yielded
3-(2-amino-3,4-dihydro-4-oxo-6,7-diphenyl-3-pteri-
dinyl)-propoxy(diisopropylamino)methoxyphosphine oxide 32
(8.8 mg, 24%), mp 123–25 ◦C (from acetone–hexane) (found:
C, 61.10; H, 6.63; N, 15.13. Calc. for C28H35N6O4P: C, 61.08;
H, 6.41; N, 15.26%); kmax (EtOH)/nm 242 inflection (log
e/dm3 mol−1 cm−1 4.42), 298 (4.50) and 382 (4.19); mmax/cm−1
3391, 3297, 3059, 1712, 1679, 1568, 1541, 1523, 1246, 1235,
1190, 1026, 1016, 838, 792 and 694; dH (200 MHz; d6-DMSO;
relative to CD2HSOCD3) 1.13 (12 H, d, J 6.6, 2 × CH(CH3)2),
1.96 (2 H, m, NCH2CH2), 3.52 (3 H, d, J 11.2, POCH3), 3.97 (2
H, m, POCH2), 4.12 (2 H, t, J 6.5, NCH2), 4.24–4.40 (2 H, m,
2 × CH(CH3)2), 7.31–7.36 (10 H, m, 6,7-diPh) and 7.65 (2 H,
br s, NH2); dC (75.5 MHz, CDCl3) 22.74, 22.53, 28.11, 40.47,
46.24, 52.67, 65.17, 125.64, 127.97, 128.09, 128.35, 129.37,
129.89, 130.18, 130.10, 138.51, 149.03, 153.40, 154.13, 158.51
and 161.31; dP (161 MHz; d6-DMSO) 14.48.
(3-(2-N,N-Dimethylaminomethyleneamino-3,4-dihydro-4-oxo-
6,7-diphenyl-3-pteridinyl)propoxy)diisopropylaminomethoxy-
phosphine oxide 33
(3-(2-Amino-3,4-dihydro-4-oxo-6,7-diphenyl-3-pteridinyl) pro-
poxy)diisopropylaminomethoxyphosphine oxide 32 (14 mg,
0.025 mmol) was dissolved in dry tetrahydrofuran (5 cm3), N,N-
dimethylformamide dimethyl acetal (7 ll, 6.3 mg, 0.053 mmol)
was added, and the solution was heated at 50 ◦C under nitrogen
for 2 h. The solution was cooled and evaporated under a reduced
pressure, when flash chromatography of the residue on silica,
eluting with a gradient of dichloromethane–ethanol (100 : 2–100
: 2.5) afforded (3-(2-N,N-dimethylaminomethyleneamino-3,4-
dihydro-4-oxo-6,7-diphenyl-3-pteridinyl)propoxy)diisopropyl-
aminomethoxyphosphine oxide 33 (10 mg, 65%), mp 128–29 ◦C
(from acetone–hexane) (found: C, 60.02; H, 6.62; N, 15.58.
Calc. for C31H40N7O4P·H2O: C, 59.70; H, 6.79; N, 15.72%); kmax
(EtOH)/nm 320 (log e/dm3 mol−1 cm−1 4.56), and 382 (4.28);
mmax/cm−1 1683, 1634, 1528, 1485, 1449, 1426, 1343, 1246,
1191, 1110, 1023, 1008, 887, 794, 770 and 695; dH (300 MHz;
CDCl3) 1.21 (12 H, d, J 6.8, 2 × CH(CH3)2), 2.14–2.20 (2 H,
m, NCH2CH2), 3.20 (3 H, s, NCH3), 3.23 (3 H, s, NCH3),
3.37–3.48 (2 H, m, 2 × CH(CH3)2, 3.65 (3 H, d, J 11.2,
POCH3), 4.03–4.17 (2 H, m, POCH2), 4.51 (2 H, t, J 7.4,
NCH2), 7.22–7.53 (10 H, m, 6,7-diPh), 8.98 (1H, s, N=CH); dP
(121.5 MHz; CDCl3) 22.41.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 3 5 8 8 – 3 6 0 1
3 5 9 9

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2-(6-Carboxypentylamino)-6-amino-5-nitroso-4-(3H)
Radiolabelling experiments
pyrimidinone 35
The 34-base oligodeoxynucleotides (ODNs) were synthesised
without a phosphate group at their 5ꢀ termini and were labelled
by the transfer of the c32P from c32P[ATP] using the enzyme
bacteriophage T4 polynucleotide kinase (PNK). The ODN
(25 pmol), radioactive isotope (c32P[ATP], 2 ll (specific activity
5000 Ci/mmol ⇒ 4pmol)), PNK buffer (10 × PNK buffer, 2 ll)
and water (9 ll) were placed in a sterile Eppendorf tube and
vortexed. The enzyme (PNK enzyme, 2 ll, 20 units) was then
added and the tube was tapped rather than vortexed to mix
the contents, since vortexing is known to denature the enzyme.
The reaction was incubated at 37 ◦C for 40 min, followed by
10 min at 68 ◦C (which inactivates the enzyme). 3M Ammonium
acetate (100 ll) and cold 98% ethanol (400 ll) were added to the
reaction mixture which was then vortexed and placed at −20 ◦C
for 20 min. The reaction tube was then centrifuged at 12 000 g
for 20 min and the supernatant was removed. Cold 98% ethanol
(1 cm3) was added and the mixture was vortexed and stored at
6-Amino-2-methylthio-5-nitroso-4-(3H)-pyrimidinone 34
(500 mg, 2.7 mmol) and 6-aminohexanoic acid (1.0 g, 7.6 mmol)
were suspended in water (20 cm3). The suspension was refluxed
for 1 h (with a hydrogen disulfide scrubber attached to the
condenser), during which time the blue suspension changed to
a red solution. The reaction was cooled in an ice bath, when
2-(6-carboxypentylamino)-6-amino-5-nitroso-4-(3H) pyrimi-
dinone 35 separated as an orange solid (300 mg, 45%), dH
(400.13 MHz; d6-DMSO) 1.3 (2 H, m, CH2), 1.52 (4 H, m,
2 × CH2), 2.21 (2 H, t, J 7.0, CH2), 7.53–8.4 (2 H, s, NH2); dH
(400.13 MHz; NaOD; Me4Si) 1.31 (2 H, br s, CH2), 1.52 (4 H,
br s, 2 × CH2), 2.14 (2 H, t, J 7.0, CH2), 3.31 (2 H, t, J 6.5, CH2);
dC (100.6 MHz; d6-DMSO) + DEPT 24.5 (CH2), 26.2 (CH2),
28.8 (CH2), 34.0 (CH2), 40.7 (CH2), 141.7 (pyrimidine C), 152.3
(pyrimidine C), 154.5 (pyrimidine C), 161.6 (pyrimidine C) and
175.0 (C=O).
◦
−20 C for 20 min. The reaction tube was then centrifuged at
12 000 g for 20 min. Following removal of the supernatant, the
pellet was dried at room temperature and stored in sterile water.
2-(6-Carboxypentylamino)-6,7-diphenyl-4-(-3H) pteridinone 36
2-(6-Carboxypentylamino)-6-amino-5-nitroso-4-(3H) pyrimi-
dinone 35 (100 mg, 0.317 mmol) was dissolved in aqueous
sodium hydroxide (2 M, 10 cm3), palladised carbon (10%,
60 mg) was added, and the mixture stirred vigorously under
hydrogen at atmospheric pressure until hydrogen uptake ceased.
The mixture was filtered under vacuum into a solution of
benzil, (78 mg, 0.32 mmol) in ethanol (10 cm3). The solution
was refluxed until TLC (dichloromethane–methanol, 15 : 2)
showed that no benzil remained. The solution was cooled to
room temperature, neutralised to pH 7 with dilute hydrochloric
acid and evaporated to dryness. The solid was extracted with
methanol and the product was purified by column chromatog-
raphy, eluting initially with dichloromethane followed by an
increasing gradient of methanol. Pure fractions were pooled
and the solvent evaporated to give 2-(6-carboxypentylamino)-
6,7-diphenyl-4-(-3H) pteridinone 36 as a yellow solid (115 mg,
73%), dH (400.13 MHz; d6-DMSO) 1.3 (2 H, m, CH2), 1.52 (4
H, m, 2 × CH2), 2.21 (2 H, t J 7.0, CH2), 3.37 (2 H, t, J 6.5,
CH2), 4.5 (2 H, br s, 2 × NH), 7.29–7.4 (10 H, m, 6,7-diPh); dC
(100.6 MHz; d6-DMSO) + DEPT 25.1 (CH2), 26.5 (CH2), 29.0
(CH2), 35.3 (CH2), 40.7 (CH2), 128.2 (C), 128.7 (C), 129.0 (C),
129.2 (C), 129.8 (C), 174.1 (C=O); m/z (ES−), 428 (M − H+).
General procedure for irradiation experiments
To an Eppendorf tube were added 1 ll of the photoactive
conjugate, 1 ll of the radiolabelled target and 8 ll of 12.5 mM
phosphate buffer/125 mM sodium chloride (pH 6.9). The tube
was vortexed to equilibrate the content◦s and centrifuged at 5000
rpm. The tube was then heated to 90 C for 5 min and cooled
slowly to room temperature to allow hybridisation to occur.
The tube was then placed in an ice water bath and irradiated
with light of wavelength >330 nm (high pressure mercury lamp
radiation passed through pyrex glass filter). After irradiation,
the sample was dried by vacuum centrifuge and 1 M piperidine
(15 ll) added. After vortexing and centrifuging, the sample was
heated at 90 ◦C for 30 min and dried by vacuum centrifuge. The
sample was washed by adding water (10 ll) and then vortexing
and centrifuging. The sample was dried by vacuum centrifuge.
Loading dye (5 ll; 1% bromophenol blue, 1% xylene cyanol in
an 80% aqueous formamide solution) was then added to the
sample prior to sequencing gel electrophoresis.
Acknowledgements
Thanks are due to Enterprise Ireland (grant SC/1998/432),
the Health Research Board of Ireland and the HEA PRTLI
(Chemical Synthesis/Chemical Biology) for financial support,
to Dr John O’Brien for NMR spectra, Dr Martin Feeney for
advice and assistance with ES MS and Dr Susan Quinn for
helpful discussions.
2-(6-Succinimidocarboxypentylamino)-6,7-diphenyl-4-(-3H)-
pteridinone 37
2-(6-Carboxypentylamino)-6,7-diphenyl-4-(3H)-pteridinone
36 (86 mg, 0.2 mmol) was suspended in anhydrous N,N-
dimethylformamide (2.5 cm3). To the suspension were added
diisopropylethylamine (61ll) and N,N,Nꢀ,Nꢀ-tetramethyl
(succinimido)uronium tetrafluoroborate (72 mg, 0.22 mmol)
and the mixture was stirred in the dark for 20 h under a
nitrogen atmosphere, when TLC (dichloromethane–methanol,
15 : 2) showed that no starting material remained. The brown
reaction mixture was evaporated to dryness under vacuum
and purified by flash chromatography, eluting initially with
dichloromethane, followed by an increasing gradient of ethanol.
Pure fractions were pooled and the solvent evaporated to
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