Nucleosides. LXV. Synthesis of New Pteridine-N8-Nucleosides

Article abstract of DOI:10.1081/NCN-120027817

A general synthetic approach to various isoxanthopterin-nucleosides starting from 6-methyl-2-methylthio-4(3H), 7(8H)-pterdinedione (1) has been developed. Ribosylation with l-O-acetyl-2,3,5-tri-O-benzoyl-β -D-ribofuranose via the silyl-method led to 2 and

Full text of DOI:10.1081/NCN-120027817

This article was downloaded by: [The University of Manchester Library]
On: 21 November 2014, At: 05:45
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
Nucleosides. LXV. Synthesis of New
Pteridine–N8–Nucleosides
Stefan Matysiak a , Bernhard Waldscheck a & Wolfgang Pfleiderer a
a Fachbereich Chemie , Universität Konstanz , Postfach 5560, 78457, Konstanz, Germany
Published online: 01 Jun 2007.
To cite this article: Stefan Matysiak , Bernhard Waldscheck & Wolfgang Pfleiderer (2004) Nucleosides. LXV. Synthesis of
New Pteridine–N8–Nucleosides , Nucleosides, Nucleotides and Nucleic Acids, 23:1-2, 51-66, DOI: 10.1081/NCN-120027817
To link to this article: http://dx.doi.org/10.1081/NCN-120027817
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 1 & 2, pp. 51–66, 2004
Nucleosides. LXV.# Synthesis of New
Pteridine–N8–Nucleosidesy
*
Stefan Matysiak, Bernhard Waldscheck, and Wolfgang Pfleiderer
Fachbereich Chemie, Universita¨t Konstanz, Konstanz, Germany
ABSTRACT
A general synthetic approach to various isoxanthopterin-nucleosides starting from 6-
methyl-2-methylthio-4(3H), 7(8H)-pterdinedione (1) has been developed. Ribosyla-
tion with 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose via the silyl-method led to
2 and reaction with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-ribofuranose using the
DBU-method afforded 28. Protection of the amide function at O4 by benzylation to 5
and by a Mitsunobu reaction with 2-(4-nitrophenyl)ethanol to 29 gave soluble
intermediates which can be oxidized to the corresponding 2-methylsulfonyl derivatives
8 and 30, respectively. Nucleophilic displacement reactions of the highly reactive
2-methylsulfonyl functions by various amines proceeded under mild conditions to
isoxanthopterin-N8-ribo- (11–17) and 2’-deoxyribomucleosides (31–33). Debenzyla-
tion can be achieve by Pd-catalyzed hydrogenation (9 to 19) and cleavage of the npe-
protecting group (31, 32 to 34, 35) works well with DBU by b-elimination.
Key Words: Pteridine nucleosides; Ribosylation reactions; 2-(4-nitrophenyl)ethyl
protecting group; Mitsunobu reaction.
#See Ref. [1].
yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.
*
Correspondence: Wolfgang Pfleiderer, Fachbereich Chemie, Universita¨t Konstanz, Postfach
5560, 78457, Konstanz, Germany; Fax: 0049-7531-883138; E-mail: wolfgang.pfleiderer@
uni-konstanz.de.
51
DOI: 10.1081/NCN-120027817
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

ORDER
REPRINTS
52
Matysiak, Waldscheck, and Pfleiderer
INTRODUCTION
Pteridine–N8–nucleosides can be regarded as homologous purine-nucleosides,
however, the enlargement of the five-membered imidazole—into the six-membered
pyrazine-ring changes the chemistry from a p-excessive into a p-deficient nitrogen-
heterocycle. Pteridine–nucleoside syntheses can be performed by various methods
including the silyl-approach,[2–4] the sodium salt method[5] and the DBU-catalyzed
glycosylation reaction,[6–8] respectively. Since substituted pteridine derivatives[9] are
fluorescent this feature is of great interest in labeling experiments especially of
oligonucleotides. A lumazine fluorophore has been incorporated into oligonucleotides
as a nucleoside analog to study in conjunction with a ruthenium complex energy
transfer interactions.[10,11] Isoxanthopterin nucleosides, in particular, show high
fluorescene quantum yields in the region of 0.9[9] and can therefore facilitate
fluorescence studies involving oligonucleotides.[12] In order to extent our knowledge
about pteridine nucleoside fluorophores new types related to isoxanthopterin-N8-ribo-
and 2’-deoxyribofuranosides have been synthesized. Since isoxanthopterin and its
derivatives are a very insoluble substances direct glycosylation reactions, in general,
have so far been without success. A valuable starting material to achieve the an-
ticipated reactions, however, has been found in the 6-methyl-2-methylthio-4(3H),
7(8H)-pteridinedione 1[7] which shows the afforded physical and chemical properties to
achieve glycosylations in organic solvents followed by chemical modifications of the
various substituents.
RESULTS AND DISCUSSION
The ribosylation of 6-methyl-2-methylthio-4(3H), 7(8H)-pteridinedione 1[7] with 1-
O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose was performed by the Birkofer–Hilbert–
Johnson silyl-method[13,14] under BF3–etherate catalysis in analogy to former
experiments[15] to give 2 in 45% yield. Treatment of 2 with methanolic ammonia at
room temp. proceeded with debenzoylation of the sugar moiety leading to 6-methyl-2-
methylthio-8-b-D-ribofuranosyl-4(3H), 7(8H)-pteridinedione 3 in good yield. In order to
improve the solubility of 2 in organic solvents the amide function was protected by
benzylation with benzyl bromide in presence of silver carbonate to give a mixture of 4-
benzyloxy-2-methylthio-8-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-7(8H)-pteridinone 4
and the corresponding 3-benzyl isomer 5 in 69% and 20% yield, respectively. 3-
Benzyl-6-methyl-2-methylthio-8-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl-4(3H), 7(8H)-
pteridinedione 5 is highly reactive towards nucleophiles at the aglycone moiety
forming 3-benzyl-6-methylisoxanthopterin-8-ribofuranoside 6 on treatment with metha-
nolic ammonia. The analogous reaction with 4 lead, expectedly, to deprotection of the
sugar benzoyl groups but additionally in this case displacement of the 4-benzyloxy
function took place yielding 4-amino-6-methyl-2-methylthio-8-b-D-ribofuranosyl-
7(8H)-pteridone 7. In order to get selective substitution in 2-position 4 was oxidized
by m-chloroperbenzoic acid to the corresponding 2-methylsulfonyl derivative 8 in 92%
yield which showed the expected reactivity due to the fact that the methylsulfonyl
function is an excellent leaving group in nucleophilic displacement reactions.
Compound 8 turned out to be a valuable intermediate for modifying the pteridine

ORDER
REPRINTS
Nucleosides. LXV
53
nucleus. Hydrolysis of 8 by 0.1 N NaOH in THF afforded 4-benzyloxy-6-methyl-
8-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-2(1H),7(8H)pteridinedione 9 whereas 1 N
NaOH in THF/H2O was associated with deblocking of the sugar moiety to give 10.
Treatment of 8 with ammonia, allylamine, n-butylamine, 2-picolylamine, dimethylamine
and morpholine in organic solvents like, dioxane, THF or AcOEt proceeded well to the 4-
benzyloxy-2-subst.amino-8-(2,3,5-tri-O-benzoyl-b-D-ribofuranosyl)-7-(8H)-pteridones
11–16. High conc. methanolic methylamine removed simultaneously the benzoyl groups
yielding 17 directly. Finally methanolate treatment of 8 afforded in one step 2,4-
dimethoxy-6-methyl-8-b-D-ribofuranosylpteridine 18. The advantage of the benzyl
protecting group is seen in its easy removal by Pd/C catalysed hydrogenolysis converting
Scheme 1.

ORDER
REPRINTS
54
Matysiak, Waldscheck, and Pfleiderer
9 into 19 followed by debenzoylation to give 6-methyl-8-b-D-ribofuranosyl-
2(1H),4(3H),7(8H)-pteridinetrione 20 (Scheme 1).
In a second series of glycosylation reactions attempts were undertaken to convert
N2,6-dimethyl- 25 and N2-b-hydroxyethyl-6-methylisoxanthopterin 27 directly with 1-
chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-ribofuranose into their 8-b-D-deoxyribofurano-
sides. The starting isoxanthopterin derivatives 25 and 27 have been synthesized from 6-
amino-2-methylthio-5-nitroso-4(3H)pyrimidone 21 by treatment with methylamine to
22 and ethanolamine to 23, respectively, followed by reduction to the corresponding 5-
amino derivatives, regioselective condensation with ethyl sodium-oxalylacetate to N2-
methyl- 24 and N2-b-hydroxyethyl-6-ethoxycarbonyl-isoxanthopterin 26 both of which
can be hydrolysed and decarboxylated to give 25 and 27, respectively. The DBU-
catalysed glycosylations in acetonitrile, however, were very unsatisfactorily since due
to the low solubility of the starting pteridines only low yields of a,b-anomeric mixtures
were obtained. From these disappointing results was realized that the anticipated
syntheses of new isoxanthopterin-deoxyribofuranoses must start from 6-methyl-2-
methylthio-8-(2-deoxy-3,5-di-O-p-toluoyl-b-D-ribofuranosyl)-4(3H),7(H)-pteridinedione
28[7] which could be prepared in improved yield of 42%. Protection of the amide
function by a Mitsunobu reaction forming the O4-2-(4-nitrophenyl)ethyl derivative 29
and its oxidation to the corresponding 2-methylsulfonyl pteridine-2’-deoxynucleoside 30
was achieved by known procedures[7] in the reported high yields. Displacement
Scheme 2.

ORDER
REPRINTS
Nucleosides. LXV
55
reactions of the methylsulfonyl group by dry methylamine and ethanolamine in CH2Cl2,
respectively, proceeded well to give 31 and 32. Deacylation of the sugar moiety in 31
works best in MeOH with KCN to form 33. Removal of the O4-2-(4-nitrophenyl)ethyl
group in 31 and 33 was performed with DBU in a b-elimination process yielding 34
and 35, respectively. Finally, it was shown that 2-b-hydroxyethylamino-6-methyl-4-2-
(4-nitrophenyl)ethoxy-8-(2-deoxy-3,5-di-O-p-toluoyl-b-D-ribofuranosyl)-7(8H)-pteridi-
none 32 can fully be protected at the b-hydroxy group by treatment with 2-(4-
nitrophenyl)ethyl chloroformate forming 36 in 88% yield (Scheme 2).
EXPERIMENTAL
All melting points are uncorrected. Products were dried under high vacuum.
TLC: precoated silica gel thin-layer sheets 60 F254 from Merck. Column chromatog-
raphy (CC): silica gel (Baker, 30–60 mm); 0.2–0.3 bar. UV/VIS: Perkin–Elmer
1
Lambda 5; lmax in nm (log e). H-NMR: Bruker AC 250; d in ppm rel. to SiMe4 or
CDCl3, (DMSO–d6) as internal standard. All solvents used were of anhydrous grade.
6-Methyl-2-methylthio-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-4(3H),7(8H)-
pteridinedione (2). A suspension of 6-methyl-2-methylthio-4(3H),7(8H)-pteridine-
dione (1)[7] (13.84 g, 62 mmol) and (NH4)2SO4 (0.25 g) in freshly destilled
hexamethyldisilazane (HMDS) (150 ml) was heated under reflux and exclusion of
moisture for 6 h. The excess of HMDS was distilled off, the yellowish residue kept
under high vacuum for 2 h and was then dissolved in CH2Cl2 (300 ml). A solution of
1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose (31.28 g, 62 mmol) in CH2Cl2 (400 ml)
was added, followed by BF3–etherate (30 ml) and then the mixture stirred at r.t. for 24
h. The reaction solution was poured slowly into a saturated solution of NaHCO3
(400 ml) (fuming!) forming an emulsion. It was stirred for 2 h and then kept in a
separation funnel over night. The organic layer was separated, dried over Na2SO4,
evaporated and coevaporated with EtOH yielding a redish amorphous foam (40.1 g).
Purification was achieved by flash silica–gel column chromatography in batches of
5.5 g with toluene/AcOEt 2:1 (600 ml), toluene/AcOEt 1:1 (500 ml), toluene/AcOEt
1:3 (500 ml) and toluene/AcOEt 1:5 (500 ml) collecting 200 ml fractions. The product
2 eluted in fractions 5–8 yielding on evaporation 2.74 g (45%) giving a total of 18.6 g.
Recrystallization of 1 g from 100 ml of EtOH gave 0.85 g of yellow-orange crystals of
mp. 232–234°C. UV (MeOH): 228 (4.68), [263, (3.96)], 282 (3.82), 298 (3.77), 344
1
(4.09), [354 (4.05)]. pKa = 8.70. H-NMR (CDCl3): 12.5 (bs, 1H, H–N(3)); 8.12–7.84
(m, 6H, arom.H); 7.60–7.27 (m, 9H, arom.H); 7.19 (d, 1H, H–C(1’)); 6.33 (pt, 1H, H–
C(3’)); 6.20 (dd, 1H, H–C(2’)); 4.92–4.64 (m, 3H, H–C(4’;5’;5@)); 2.51 (s, 3H, Me–S);
2.39 (s, 3H, Me(6).
Anal. For C34H28N4O9S (668.6) Calcd.: C, 61.08; H, 4.22; N, 8.38. Found: C,
60.98; H, 4.23; N, 8.43.
6-Methyl-2-methylthio-8- -D-ribofuranosyl-4(3H),7(8H)-pteridinedione (3). In
saturated methanolic ammonia (50 ml) compound 2 (1.2 g, 1.8 mmol) was stirred at r.t.
for 72 h. It was evaporated to dryness, the residue dissolve in H2O (150 ml), acidified
with AcOH and then extracted with AcOEt (4 ꢀ 50 ml). The H2O layer was

ORDER
REPRINTS
56
Matysiak, Waldscheck, and Pfleiderer
concentrated till a precipitate starts to separate. The colorless crystals were collected
after standing over night, washed with little cold H2O and dried in high vacuum to give
0.487 g (76%) of mp. 235°C (decomp.). UV (pH 4): 214 (4.43), [244, 399)], 261
1
(4.09), 351 (4.15). pKa = 6.55. H-NMR (DMSO–d6): 13.16 (bs, 1H, H–N(3)); 6.73
(d, 1H, H–C(1’)); 4.56 (dd, 1H, H–C(2’)); 4.21 (pt, 1H, H–C(3’)); 3.79–3.37 (m, 3H,
H–C(4’;5’;5@)); 2.54 (s, 3H, Me–S); 2.28 (s, 3H, Me(6).
Anal. For C13H16N4O6S (356.6) Calcd.: C, 43.82; H, 4.53; N, 15.72. Found: C,
44.03; H, 4.58; N, 15.66.
3-Benzyl-6-methyl-2-methylthio-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-
4(3H),7(8H)-pteridinedione (4) and 4-benzyloxy-6-methyl-2-methylthio-8-(2,3,5-tri-
O-benzoyl- -D-ribofuranosyl)-4(3H),7(8H)-pteridinedione (5). A suspension of 2
(6.05 g (9 mmol) and silver carbonate (6.34 g) in dry benzene (110 ml) was heated
with stirring to 80°C for 2 h. After cooling to 60°C benzylbromide (4.5 ml, 36 mmol)
was added and the mixture stirred at 60–70°C for 48 h. The reaction solution was
filtered hot through a thin layer of SiO2, washed several times with toluene and the
filtrates concentrated to 20 ml for CC (SiO2, 220 g, 250 ml fractions) with each 500 ml
of toluene/AcOEt 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1 and 2:1. Fraction 9–
13 gave after evaporation 4.71 g (69%) of 5 and fractions 17–20 1.38 g (20%) of 4.
4: Recrystallization from EtOH (80 ml) gave 0.956 g (14%) of a colorless crystal
powder of mp. 108–109°C. UV (MeOH): 227 (4.78), 282 (4.61), 301 (3.91), 343
1
(4.15), [353 (4.06)]. H-NMR (CDCl3): 8.12–7.26 (m, 20H, arom.H); 7.16 (d, 1H, H–
C(1’)); 6.35 (pt, 1H, H–C(3’)); 6.19 (dd, 1H, H–C(2’)); 5.50 (d, 1H, CH2); 5.26 (d, 1H,
CH2); 4.90–4.63 (m, 3H, H–C(4’;5’;5@)); 2.54 (s, 3H, Me–S); 2.34 (s, 3H, Me(6).
Anal. For C41H34N4O9S (758.8) Calcd.: C, 64.90; H, 4.52; N, 7.38. Found: C,
64.89; H, 4.45; N, 7.24.
5: Recrystallization from EtOH (180 ml) gave 4.13 g (61%) of a colorless crystal
powder of mp. 89–91°C. UV (MeOH): 224 (4.72), 275, (3.84), 282 (3.86), 328 (4.19),
333 (4.20), [346 (4.11)]. H-NMR (DMSO–d6): 7.98–7.79 (m, 5H, arom.H); 7.69–
1
7.32 (m, 15H, arom.H); 7.18 (d, 1H, H–C(1’)); 6.34 (pt, 1H, H–C(3’)); 6.18 (dd, 1H,
H–C(2’)); 5.65–5.54 (dd, 2H, CH2); 4.83–4.52 (m, 3H, H–C(4’;5’;5@)); 2.51 (s, 3H,
Me–S); 2.38 (s, 3H, Me(6).
Anal. For C41H34N4O9S (758.8) Calcd.: C, 64.90; H, 4.52; N, 7.38. Found: C,
64.97; H, 4.54; N, 7.31.
3-Benzyl-6-methyl-8- -D-ribofuranosyl-isoxanthopterin (6). A suspension of 4
(1.19 g, 1.5 mmol) in saturated methanolic ammonia (30 ml) was stirred at r.t. for 5
days under exclusion of light to give an orange colored solution. It was evaporated to
dryness, the residue treated in toluene/AcOEt 10:1 by ultrasound and then the solid
(0.5 g) collected. Recrystallization from H2O (7 ml) with charcoal gave 0.372 g
(59%) of a colorless crystal powder of mp. 154°C (decomp.). UV (MeOH): 217
(4.56), [257 (3.54)], 297 (3.98), 346 (4.11). 1H-NMR (DMSO–d6): 7.77 (bs, 2H,
NH2); 7.40–7.15 (m, 5H, arom. H); 6.62 (d, 1H, H–C(1’)); 5.28–5.12 (2d, 2H, CH2);
4.62 (dd, 1H, H–C(2’)); 4.25 (pt, 1H, H–C(3’)); 3.73–3.41 (m, 3H, H–C(4’;5’;5@));
2.23 (s, 3H, Me(6).
Anal. For C19H21N5O6 (415.4) Calcd.: C, 54.94; H, 5.10; N, 16.86. Found: C,
54.68; H, 5.41; N, 16.85.

ORDER
REPRINTS
Nucleosides. LXV
4-Amino-6-methyl-2-methylthio-8- -D-ribofuranosyl-7(8H)-pteridinone (7).
57
A
suspension of 5 (0.758 g, 1 mmol) in saturated methanolic ammonia (20 ml) was stirred
at r.t. for 3 days. The resulting solution was evaporated, the residue was treated with cold
MeOH (5 ml), filtered and dried to give 0.247 g (69%). Recrystallization from EtOH
gave 0.167 g (47%) of a crystal powder of mp. 242°C (decomp.). UV (MeOH): 216
1
(4.42), [248 (4.05)], 264 (4.16), [292 (3.65)], 346 (4.13). H-NMR (DMSO–d6): 7.77
(bs, 1H, NH2); 7.58 (bs, 1H, NH2); 6.67 (d, 1H, H–C(1’)); 4.59 (dd, 1H, H–C(2’)); 4.23
(pt, 1H, H–C(3’)); 3.78–3.47 (m, 3H, H–C(4’;5’;5@)); 2.32 (s, 3H, Me(6).
Anal. For C13H17N5O5S (355.4) Calcd.: C, 43.94; H, 4.82; N, 19.71. Found: C,
44.10; H, 4.88; N, 19.38.
4-Benzyloxy-6-methyl-2-methylsulfonyl-8-(2,3,5-tri-O-benzoyl- -D-ribofurano-
syl)-7(8H)-pteridinone (8). To a solution of 5 (10.5 g, 13.8 mmol) in CH2Cl2
(350 ml) was added in small portions m-chloroperbenzoic acid (8.25 g containing 15%
chlorobenzoic acid) with stirring. After 12 h the precipitated m-chlorobenzoic acid was
filtered off, the filtrate concentrated to 50 ml and then put onto a silica–gel column
(240 g SiO2, 250 ml fractions) for chromatography with toluene/AcOEt 100:1 (1 l),
50:1 (1 l), 30.1 (600 ml), 20:1 (600 ml), 10:1 (600 ml), 7:1–3:1 (2 l). The product
fractions 13–19 were evaporated, the residue coevaporated with CH2Cl2 (3 ꢀ 100 ml)
and then dried in high vacuum to give 10.2 g (92%) of a colorless solid foam. UV
(MeOH): [217 (4.64)], 231 (4.76), [276 (3.90)], 282 (3.94), 307 (4.08), [314 (4.05)].
1H-NMR (DMSO–d6): 7.98–7.79 (m, 5H, arom.H); 7.69–7.32 (m, 15H, arom.H); 7.18
(d, 1H, H–C(1’)); 6.34 (pt, 1H, H–C(3’)); 6.18 (dd, 1H, H–C(2’)); 5.65–5.54 (dd, 2H,
CH2); 4.83–4.52 (m, 3H, H–C(4’;5’;5@)); 2.51 (s, 3H, Me–S); 2.38 (s, 3H, Me(6)).
Anal. For C41H34N4O11S (790.8) Calcd.: C, 62.27; H, 4.33; N, 7.09. Found: C,
61.75; H, 4.28; N, 6.91.
4-Benzyloxy-6-methyl-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-2(1H),7(8H)-
pteridinedione (9). To a stirred solution of 8 (0.84 g, 1.06 mmol) in THF (24 ml)
was added very slowly dropwise in 6 h 0.1 N NaOH (24 ml). The emulsion was
concentrated in vacuum to 20 ml, then diluted with H2O (30 ml) and treated with
Lewatit ion-exchange-resin (H+ form) to pH 4–5. The solution was extracted with
AcOEt (4 ꢀ 50 ml), the H2O layer evaporated and the residue purified by CC with a
gradient of toluene/AcOEt 50:1–5:1. The product fraction was evaporated to give 0.58 g
of a solid foam. Further purification was done on preparative silica–gel plates by
repeated chromatography with CHCl3/MeOH 100:1 (3 ꢀ) and CHCl3/MeOH 80:1
(1 ꢀ). The main band was eluted with AcOEt, evaporated and coevaporated with
CH2Cl2 to give 0.22 g (28%) of mp. 161°C. UV (pH 5): 206 (4.76), 232 (4.61), [274,
1
(3.97)], 327 (4.01), [342 (3.93)]. H-NMR (CDCl3): 8.05–7.82 (m, 5H, arom.H); 7.55–
7.20 (m, 15H, arom.H); 6.93 (s, 1H, H–C(1’)); 6.52 (bs, 1H, H–C(3’)); 6.33 (bs, 1H,
H–C(2’)); 5.50 (dd, 2H, CH2); 4.90–4.52 (m, 3H, H–C(4’;5’;5@)); 2.50 (s, 3H, Me(6)).
Anal. For C40H32N4O10 (728.7) Calcd.: C, 65.93; H, 4.43; N, 7.69. Found: C,
65.58; H, 4.49; N, 7.42.
4-Benzyloxy-6-methyl-8- -D-ribofuranosyl-2(1H),7(8H)-pteridinedione (10).
To a solution of 8 (1.0 g, 1.26 mmol) in THF (60 mnl) and H2O (30 ml) was
added slowly dropwise with stirring 1 N NaOH (10 ml). After stirring at r.t. for 24 h

ORDER
REPRINTS
58
Matysiak, Waldscheck, and Pfleiderer
the reaction solution was slightly acidified with AcOH, the THF was distilled off,
H2O (30 ml) was added and then nextracted with AcOEt (4 ꢀ 30 ml). The organic
layer was separated, washed with H2O, dried over Na2SO4, concentrated to 5 ml,
cooled over night and then the crystals collected. After drying in high vacuum 0.205
g (39%) colorless crystals of mp. 140°C were obtained. UV (pH 7): 205 (4.59), [228
(4.07)], 258 (3.76), 280 (3.76), 353 (4.18). pKa 4.76. 1H-NMR (DMSO–d6): 12.56
(bs, 1H, H–N); 7.52–7.32 (m, 5H, arom. H); 6.63 (d, 1H, H–C(1’)); 5.51 (2d, 2H,
CH2); 4.64 (dd, 1H, H–C(2’)); 4.24 (pt, 1H, H–C(3’)); 3.77–3.45 (m, 3H, H–
C(4’;5’;5@)); 2.28 (s, 3H, Me(6).
Anal. For C19H20N4O7 (416.4) Calcd.: C, 54.81; H, 4.84; N, 13.46. Found: C,
55.06; H, 4.93; N, 13.16.
2-Amino-4-benzyloxy-6-methyl-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-
7(8H)-pteridinone (11). In a solution of dioxane (6 ml), saturated with ammonia,
8 (0.2 g, 0.25 mmol) was stirred for 80 min. It was evaporated, the residue dissolved in
AcOEt (30 ml) and wahed with H2O. The organic phase was dried over Na2SO4,
evaporated, the residue dissolved in MeOH, treated with charcoal, concentrated to 5 ml
and then H2O dropwise added till a colorless precipitate starts to separate. After
standing in thev icebox over night, the crystals were collected, dried in high vacuum to
give 0.142 g (78%) of mp. 103°C. UV (MeOH): 207 (4.69), 229 (4.70), 274 (3.87), 282
1
(3.91), 288 (3.83), 345 (4.16). H-NMR (DMSO–d6): 7.68–7.34 (m, 22H, arom. H,
NH2); 7.13 (bs, 1H, H–C(1’)); 6.36 (pt, 1H, H–C(3’)); 6.24 (bs, 1H, H–C(2’)); 5.48
(2d, 2H, CH2); 4.73–4.51 (m, 3H, H–C(4’;5’;5@)); 2.28 (s, 3H, Me(6).
Anal. For C40H33N5O9 (727.7) Calcd.: C, 66.02; H, 4.57; N, 9.62. Found: C, 65.03;
H, 4.62; N, 9.39.
2-Allylamino-4-benzyloxy-6-methyl-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-
7(8H)-pteridinone (12). A solution of 8 (0.2 g, 0.25 mmol) in THF (10 ml) was
treated with allylamine (45 mg, 0.8 mmol) by stirring at r.t. for 30 min. It was
evaporated, the residue dissolved in AcOEt (40 ml), washed with H2O, the organic
layer dried over Na2SO4 and again evaporated to an oil. It was dissolved in EtOH
(15 ml), treated with charcoal, filtered and to the hot solution H2O (15 ml) added. After
cooling the crystals were collected, dried in high vacuum to give 0.108 g (56%) of mp.
161–162°C. UV (MeOH): 209 (4.66), 230 (4.71), [274 (3.82)], 282 (3.86), [293 (3.79)],
1
356 (4.21). H-NMR (DMSO–d6): 8.01–7.35 (m, 21H, arom. H, NH); 7.18 (bd, 1H,
H–C(1’)); 6.41–6.24 (m, 2H, H–C(2’), H–C(3’)); 5.80 (m, 1H, CH2 CH–); 5.50 (d,
2H, CH2); 5.05 (m, 2H, CH2 CH); 4.78–4.58 (m, 3H, H–C(4’;5’;5@)); 3.93 (bs, 2H,
CH2 CHCH2); 2.28 (s, 3H, Me(6).
Anal. For C43H37N5O9 (767.8) Calcd.: C, 67.28; H, 4.86; N, 9.12. Found: C, 67.43;
H, 4.86; N, 9.04.
4-Benzyloxy-2-n-butylamino-6-methyl-8-(2,3,5-tri-O-benzoyl- -D-ribofurano-
syl)-7(8H)-pteridinone (13). Analogous to the preceeding procedure with 8 (0.2 g,
0.25 mmol) and n-butylamine (73 mg, 1 mmol) in 45 min. The same work-up gave
0.114 g (59%) of colorless crystals of mp. 164°C. UV (MeOH): 209 (4.68), 230 (4.71),
[274 (3.82)], 282 (3.88), [292 (3.80)], 352 (4.21). 1H-NMR (DMSO–d6): 8.09–7.36 (m,
21H, arom. H, NH); 7.18 (bd, 1H, H–C(1’)); 6.41–6.24 (m, 2H, H–C(2’), H–C(3’));

ORDER
REPRINTS
Nucleosides. LXV
59
5.50 (2d, 2H, CH2); 4.74–4.52 (m, 3H, H–C(4’;5’;5@)); 3.83 (m, 2H, HNCH2); 2.28 (s,
3H, Me(6); 1.42 (m, 2H, CH2); 1.05 (Mm, 2H, CH2); 0.81 (t, 3H, CH3).
Anal. For C44H41N5O9 (783.8) Calcd.: C, 67.42; H, 5.23; N, 8.94. Found: C, 67.55;
H, 5.27; N, 8.81.
4-Benzyloxy-6-methyl-2-(2-picolylamino)-8-(2,3,5-tri-O-benzoyl- -D-ribofura-
nosyl)-7(8H)-pteridinone (14). Analogous to the preceeding procedure with 8 (0.2 g,
0.25 mmol) in AcOEt (20 ml) and 2-picolylamine (0.1 g, 0.92 mmol) in 45 min. The
same work-up gave 0.182 g (89%) of colorless crystals of mp. 144°C. UV (MeOH):
1
230 (4.72), [262 (4.02)], 282 (3.92), [290 (3.85)], 348 (4.23). H-NMR (DMSO–d6):
8.48 (d, 1H, a-pyridyl); 7.94–7.14 (m, 24H, arom. H, NH); 6.92 (bd, 1H, H–C(1’));
6.35–6.19 (m, 2H, H–C(2’), H–C(3’)); 5.50 (s, 2H, OCH2); 5.34 (s, 2H, N–CH2);
4.70–4.51 (m, 3H, H–C(4’;5’;5@)); 2.23 (s, 3H, Me(6)).
Anal. For C46H28N6O9 (818.8) Calcd.: C, 67.47; H, 4.68; N, 10.26. Found: C,
67.70; H, 4.66; N, 10.29.
4-Benzyloxy-6-methyl-2-dimethylamino-8-(2,3,5-tri-O-benzoyl- -D-ribofurano-
syl)-7(8H)-pteridinone (15). A solution of 8 (0.37 g, 0.47 mmol) in toluene (10 ml)
was treated at r.t. with a saturated solution of dimethylamine in benzene (1 ml) for
30 min with stirring. It was dilute with toluene (20 ml), washed with H2O (3 ꢀ 10 ml),
the organic phasae dried over NA2SO4 and then evaporated. The residue was
recrystallized from EtOH to give 0.238 g (66%) of colorless crystals of mp. 124°C. UV
1
(MeOH): 212 (4.66), 229 (4.71), 282 (3.82), 295 (3.78), 358 (4.23). H-NMR (DMSO–
d6): 8.10–7.79 (m, 6H, arom. H); 7.65–7.55 (m. 3H, arom. H); 7.48–7.30 (m, 11H,
arom. H); 7.20 (bs, 1H, H–C(1’)); 6.28 (m, 2H, H–C(2’), H–C(3’)); 5.54 (q, 2H,
OCH2); 4.70–4.56 (m, 3H, H–C(4’;5’;5@)); 3.00 (s, 6H, Me2N); 2.30 (s, 3H, Me(6)).
Anal. For C46H38N5O9 (756.8) Calcd.: C, 66.67; H, 5.06; N, 9.26. Found: C, 66.92; H,
4.96; N, 9.14.
4-Benzyloxy-6-methyl-2-morpholino-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-
7(8H)-pteridinone (16). A solution of 8 (0.2 g, 0.25 mmol) in THF (10 ml) was
treated with morpholine (90 mg, 1 mmol) and stirring for 45 min at r.t. It was dilute
with AcOEt (40 ml), washed with H2O (3 ꢀ 20 ml), the organic phasae dried over
Na2SO4 and then evaporated. The residue was recrystallized from EtOH/H2O to give
0.117 g (63%) of colorless crystals of mp. 167°C. UV (MeOH): 212 (4.70), 229 (4.71),
1
274 (3.82), 294 (3.81), 354 (4.24). H-NMR (DMSO–d6): 8.00–7.30 (m, 20H, arom.
H); 7.19 (s, 1H, H–C(1’)); 6.27–6.15 (m, 2H, H–C(2’), H–C(3’)); 5.58–5.45 (2d, 2H,
OCH2); 4.77–4.52 (m, 3H, H–C(4’;5’;5@)); 3.54 (m, 4H, morpholino); 3.71 (m, 4H,
morpholino); 2.30 (s, 3H, Me(6)).
Anal. For C44H39N5O10 (738.8) Calcd.: C, 66.24; H, 4.94; N, 8.78. Found: C,
66.40; H, 4.93; N, 8.43.
4-Benzyloxy-6-methyl-2-methylamino-8- -D-ribofuranosyl-7(8H)-pteridinone
(17). A solution of 8 (0.2 g (0.25 mmol) in MeOH (10 ml) was treated with methanolic
methylamine (44%, 1 ml) and stirred at r.t. for 60 h. The resulting precipitate was
collected, washed with MeOH and dried in high vacuum to give 48 mg (45%) of a
colorless powder of mp. 225°C. UV (MeOH): 211 (4.54), 238 (4.20), [287 (3.76)], 293

ORDER
REPRINTS
60
Matysiak, Waldscheck, and Pfleiderer
(3.78), 352 (4.21). 1H-NMR (DMSO–d6): 7.46–7.30 (m, 5H, arom. H); 6.64 (bs, 1H, H–
C(1’)); 5.51 (d, 1H, CH2); 5.43 (d, 1H, CH2); 4.61 (bs, 1H, H–C(2’)); 4.25 (bs, 1H, H–
C(3’)); 3.72–3.39 (m, 3H, H–C(4’;5’;5@)); 2.83 (bs, 3H, Me–N); 2.23 (s, 3H, Me(6)).
Anal. For C20H23N5O6 (429.4) Calcd.: C, 55.94; H, 5.45; N, 16.48. Found: C,
55.94; H, 5.50; N, 16.43.
2,4-Dimethoxy-6-methyl-8- -D-ribofuranosyl-7(8H)-pteridinone (18). Com-
pound 8 (0.4 g, 5 mmol) was stirred in 0.2 N CH3ONa solution (10 ml) at r.t. for 2
days. Then H2O (20 ml) was added and then neutralized with Lewatit ion-exchange-
resin (H+ form). It was evaporated, the residue dissolved in little MeOH and put onto
2 preparative silica–gel plates for chromatography with AcOEt/MeOH 30:1 (2
developments). The main bands have been eluted with AcOEt (100 ml), evaporated and
the residue dried in high vaccum to give 32 mg (18%) of a colorless crystal powder of
mp. 193°C. UV (pH 7): 236 (3.98), [244 (3.94)], [276 (3.78)], 313 (4.13)], [321 (4.10)],
1
[339 (3.87)]. H-NMR (DMSO–d6): 6.62 (d, 1H, H–C(1’)); 4.63 (dd, 1H, H–C(2’));
4.26 (pt, 1H, H–C(3’)); 4.03 (s, 3H, MeO); 3.96 (s, 3H, MeO); 3.79–3.37 (m, 3H, H–
C(4’;5’;5@)); 2.33 (s, 3H, Me(6)).
Anal. For C18H20N4O7 (354.3) Calcd.: C, 47.46; H, 5.12; N, 15.81. Found: C,
47.41; H, 5.21; N, 15.75.
6-Methyl-8-(2,3,5-tri-O-benzoyl- -D-ribofuranosyl)-2(H),4(3H,7(8H)-pteridine-
trione (19).[16] A solution of 9 (0.7.3 g, 1 mmol) in AcOEt (30 ml) was hydrogenated
with H2 in presence of Pd/C catalyst (0.25 g, 5%) in a shaking apparatus till the uptake
of H2 is finished after 2 days. The mixture was heated, filtered hot and then the filtrate
evaporated to dryness. The residue was purified by CC (15 g, SiO2) with 200 ml of
each toluene/AcOEt 2:1, 1:1 followed by AcOEt/MeOH 25:1, 10:1 and 5:1. The
product fraction was evaporated and the residue recrystallized from EtOH (121 ml) to
give 0.372 g (58%) of mp.>186°C (decomp.). UV (pH 5): 230 (4.57), 285, (3.95), 353
1
(3.94); (MeOH): 229 (4.64), 283 (4.02), 290 (4.02), 356 (4.06). pKa 2.14, 13.12. H-
NMR (DMSO–d6): 11.17 (bs, 1H, H-N); 8.02–7.37 (m, 15H, arom. H); 6.75 (s, 1H,
H–C(1’)); 6.25 (pt, 1H, H–C(3’)); 6.10 (bs, 1H, H–C(2’)); 4.70–4.52 (m, 3H, H–
C(4’;5’;5@)); 2.22 (s, 3H, Me(6)).
Anal. For C38H26N4O10 ꢀ H2O (656.6) Calcd.: C, 60.37; H, 4.30; N, 8.53. Found:
C, 60.34; H, 4.35; N, 8.26.
6-Methyl-8- -D-ribofuranosyl-2(1H),4(3H),7(8H)-pteridinetrione (20).[16]
A
solution of 19 (0.24 g, 0.4 mmol) in saturated methanolic ammonia (20 ml) was stirred
at r.t. in the dark for 6 days. It was evaporated to dryness, the residue treated with
MeOH, the solid collected and dried in high vacuum to give 46 mg (32%) a colorless
crystal powder of mp.> 150°C (decomp.). UV (pH 6): 209 (4.38), 291, (3.93), 342
(3.98). pKa 3.42, 13.11. 1H-NMR (DMSO–d6): 6.75 (s, 1H, H–C(1’)); 4.62 (dd, 1H, H–
C(2’)); 4.25 (pt, 1H, H–C(3’)); 3.76–3.41 (m, 3H, H–C(4’;5’;5@)); 2.13 (s, 3H, Me(6)).
Anal. For [C12H13N4O7] NH4 (343.3) Calcd.: C, 41.98; H, 4.99. Found: C, 41.86;
H, 4.76.
6-Amino-2-methylamino-5-nitroso-4(3)-pyrimidone (22). A suspension of 6-
amino-2-methylthio-5-nitroso-4(3H)pyrimidone (21)[17] (8.0 g, 43 mmol) in 20%

ORDER
REPRINTS
Nucleosides. LXV
61
aqueous methylamine (120 ml) was stirred at 5–10°C for 6 h. The violet suspension is
converted into a dark-red solution. It was evaporated to dryness, the residue was
suspended in H2O (200 ml), slightly acidified by AcOH to pH 4 to get an orange
colored precipitate. The solid was collected, washed with cold H2O and acetone, to
give after drying 6.9 g (95%) of orange powder. A sample (0.15 g) was recrystallized
from H2O (200 ml) with charcoal to give 0.104 g (68%) orange glittering crystals of
1
mp.>350°C. UV (pH 12): 208 (4.01), [226 (3.57)], [264 (3.57)], 325 (4.35). H-NMR
(DMSO–d6): 11.24 (bs, 1H, H–N(3)); 10.93 (bs, 1H, H–N(6)); 8.52 (bs, 1H, H–N(6));
7.23 (bs, 1H, MeN–H); 2.85 (d, 3H, MeN).
Calc. For C5H7N5O2 (169.2): C, 35.50, H, 4.18, N, 41.42. Found: C, 35.52, H,
4.29, N, 41.81.
6-Amino-2-(2-hydroxyethyl)amino-5-nitroso-4(3H)-pyrimidone (23). A sus-
pension of 21 (7.0 g, 38 mmol) in a mixture of ethanolamine (40 ml) and H2O
(50 ml) was stirred at r.t. for 24 h. The solution was cooled and then acidified by
dropwise addition of AcOH (100ml). The resulting precipitate was collected, washed
with H2O, EtOH and ether to give after drying 6.1 g (81%). Purification was achieved
by dissolving in 3 N NaOH (20 ml), charcoal treatment, filtration and dropwise addition
of the filtrate into hot AcOH (35 ml) to form an orange-yellow solid 4.6 g (61%). A
sample (0.15 g) was recrystallyzed from H2O (200 ml) to give 0.1 g of orange crystals
of mp.> 350°C. UV (pH 12): 207 (4.01), [227 (3.50)], [264 (3.53)], 326 (4.37). 1H-NMR
(DMSO–d6):11.22 (bs, 1H, H–N(3)); 10.60 (bs, 1H, H–N(6)); 8.50 (bs, 1H, H–N(6));
7.38 (bs, 1H, H–NCH2CH2); 4.94 (t, 2H, CH2CH2OH); 3.54–3.37 (m, 4H, CH2CH2).
Calc. For C6H9N5O3 (199.2): C, 36.18, H, 4.55, N, 35.17. Found: C, 36.11, H,
4.73, N, 34.88.
6-Ethoxycarbonylmethyl-2-methylamino-4(3H),7(8H)-pteridinedione (24).
A
20% ammonium sulfide solution (32 ml) was heated to 70°C and then under stirring 22
(8.0 g, 47 mmol) added. The color of the solution turned yellow and a yellow precipitate
separated out. It was heated 30 min at 50°C, then cooled in the icebox and the solid
collected after a few hours. The yellow material was washed with cold H2O and EtOH and
dried in high vacuum to give 7.2 g (98%) of 5,6-diamino-2-methylamino-4(3H)-
pyrimidone of mp.> 300°C (decomp.). A mixture of 5,6-diamino-2-methylamino-4(3H)-
pyrimidone (7.0 g, 45 mmol) and sodium diethyl oxalylacetate (9.7 g, 46 mmol) was
heated in AcOH (275 ml) under reflux for 45 min. A yellow precipitate separated from the
hot solution. After cooling the solid was collected, washed with H2O and EtOH and dried
to give 10.5 g (83%) of chromatographically pure 24. A sample (0.25 g) was recrystallized
from H2O with charcoal to give 0.166 g of colorless fine needles of mp.> 350°C. UV
1
(MeOH): 215 (4.50), [234 (3.99)], 292 (4.06), 342 (4.21). H-NMR (DMSO–d6): 12.45
(bs, 1H, H–N(8)); 11.13 (bs, 1H, H–N(3)); 6.82 (bs, 1H, MeN–H); 4.07 (q, 2H,
OCH2CH3); 3.61 (s, 2H, CH2COOEt); 2.84 (d, 3H, N–CH3); 1.16 (t, 3H, OCH2CH3).
Calc. For C11H13N5O4 (279.3): C, 47.31, H, 4.70, N, 25.09. Found: C, 46.94, H,
4.66, N, 24.71.
6-Methyl-2-methylamino-4(3H),7(8)-pteridinedione (25). A solution of 24
(5.1 g, 18 mmol) in 1 N NaOH (200 ml) was heated under reflux for 30 min, then
treated with charcoal and the hot filtrate added dropwise into boiling AcOH (100 ml).

ORDER
REPRINTS
62
Matysiak, Waldscheck, and Pfleiderer
The resulting yellow precipitate was collected after cooling, washed with H2O and
dried at 100°C to give 3.5 g (92%) of 25. Further purification was achieved by
reprecipitation. The solid was dissolved in dilute NaOH (150 ml), again treatment with
charcoal and the filtrate added nslowly into a boiling mixture of AcOH (50 ml) and
H2O (50 ml) yielding after drying 3.4 g (89%) of a colorless crystal powder of
1
mp.> 350°C. UV (pH 12): 221 (4.59), [247 (4.19)], [279 (3.85)], 342 (4.20). H-NMR
(DMSO–d6): 12.23 (bs, 1H, H–N(8)); 11.01 (bs, 1H, H–N(3)); 6.75 (bs, 1H, MeN–
H); 2.82 (d, 3H, N–CH3); 2.21 (s, 3H, CH3–C(6)).
Calc. For C8H9N5O2 (207.2): C, 46.37, H, 4.39, N, 33.81. Found: C, 46.41, H,
4.32, N, 33.51.
6-Ethoxycarbonylmethyl-2-(2-hydroxyethyl)amino-4(3H),7(8H)-pteridinedione
(26). To a ice-cold solution of 23 (3.4 g, 17 mmol) in 1 N NaOH (25 ml) was added
hydrazine hydrate (0.9 g) and Ni–Al alloy (0.55 g). After a few min a vigorous
reaction takes place and a color change from red to yellow is observed. It was stirred at
r.t. for 1 h, the Raney-nickel filtered off and then the cooled filtrate neutralized with
HCl (20%), EtOH (10 ml) added and stored in the icebox for 2 days. The yellowish
precipitate was collected, washed with cold EtOH and ether and dried in a vacuum
desiccator to give 3.1 g (98%) of 5,6-diamino-2-(2-hydroxyethyl)amino-4(3H)-
pyrimidone of mp.> 200°C. This substance (5.4 g, 29 mmol) was heated with sodium
diethyl oxalylacetate (6.3 g, 30 mmol) in conc. AcOH (50 ml) to 80°C for 1 h. The
resulting yellow precipitate was collected after cooling, washed with H2O and EtOH
and dried at 100°C to give 5.5 g (61%) of mp.> 350°C. A small sample (0.16 g) was
recrystallized from H2O (45 ml) with charcoal to give sligthly yellowish fine needles.
1
UV (MeOH): 216 (4.49), [234 (3.99)], 292 (4.06), 342 (4.22). H-NMR (DMSO–d6):
12.45 (bs, 1H, H–N(8)); 10.86 (bs, 1H, H–N(3)); 6.99 (bs, 1H, MeN–H); 4.93 (t, 1H,
OH); 4.07 (q, 2H, OCH2CH3); 3.61 (s, 2H, CH2COOEt); 3.55–3.38 (m, 4H, CH2CH2);
1.17 (t, 3H, OCH2CH3).
Calc. For C12H15N5O5 (309.3): C, 46.60, H, 4.90, N, 22.65. Found: C, 46.66, H,
4.95, N, 22.71.
6-Methyl-2-(2-hydroxyethyl)amino-4(3H),7(8)-pteridinedione (27). A solution
of 26 (3.0 g, 9.7 mmol) in 1 N NaOH (30 ml) was heated under reflux for 30 min.
After treatment with charcoal and filtration the hot filtrate was added dropwise with
stirring into AcOH (30 ml) forming a yellow precipitate which was collected after
cooling, washed with H2O and EtOH, dried at 100°C to give 2.1 g (91%) of a yellow
powder of mp.>350°C. A sample (0.285 g) was recrystallized from H2O (400 ml) to
give 0.176 g of yellowish glittering crystals. UV (pH 12): 222 (4.52), [249 (4.15)],
1
[276 (3.87)], 340 (4.17). H-NMR (DMSO–d6): 12.26 (bs, 1H, H–N(8)); 10.78 (bs, 1H,
H–N(3)); 6.91 (bs, 1H, H–NCH2CH2); 4.92 (bs, 1H, HO–CH2); 3.55–3.35 (m, 4H,
CH2CH2); 2.21 (d, 3H, CH3–C(6).
Calc. For C9H11N5O3 ꢀ 0.5 H2O (246.2): C, 43.91, H, 4.91, N, 28.45. Found: C,
44.00, H, 4.81, N, 28.69.
6-Methyl-2-methylthio-8-(2-deoxy-3,5-di-O-p-toluoyl- -D-ribofuranosyl)-
4(3H),7(8H)-pteridinedione (28).[7] A suspension of 1 (0.2 g, 0.9 mmol) in dry
acetonitrile (30 ml) was treated with DBU (0.4 g, 2.65 mmol) and stirred at r.t. till a

ORDER
REPRINTS
Nucleosides. LXV
63
clear solution (30 min) was obtained. The 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-a-D-
ribofuranose (0.7 g, 1.8 mmol) was added and the mixture stirred for 30 h. It was
neutralized with AcOH in CH2Cl2 and then evaporated to dryness. The dark-colored
residue was purified by disssolving in CH2Cl2 and CC (SiO2) with toluene/AcOEt 1:1
(150 ml), 1:2 (200 ml) and CH2Cl2/MeOH 40:1 (200 ml). The product fraction were
evaporated to give 0.265 g (52%) of an a/b-anomeric mixture. Recrystallization from
tolene gave 0.213 g (42%) of pure b-anomer as colorless crystals of mp. 196°C. Lit.[7]
mp. 196–197°C.
6-Methyl-2-methylamino-8-(2-deoxy-3,5-di-O-p-toluoyl- -D-ribofuranosyl)-O4-
[2-(4-nitrophenyl)ethyl]-7(8H)-pteridinone (31). Through a solution of 30[7] (1.0 g,
1.32 mmol) in dry CH2Cl2 (90 ml) was bubbled a dry stream of methylamine for
1 h with stirring. It was then evaporated to dryness and coevaporated with CH2Cl2.
Purification was done by CC with toluene/AcOEt 5:2. The product fraction was again
evaporated to give a yellowish solid foam. Recrystallization from EtOH yielded 0.607 g
(65%) colorless crystals of mp. 174°C. UV (MeOH): 202 (4.83), [214 (4.61)], 239
1
(4.72), [272 (4.28)], 349 (4.27). H-NMR (CDCl3): 8.20 (d, 2H, o to NO2); 8.05–7.85
(m, 4H, arom. H); 7.50 (d, 2H, m to NO2); 7.36 (m, 1H, H–C(1’)); 7.26–7.13 (2d, 4H,
o to CH3); 6.06 (m, 1H, H–C(3’)); 5.72 (bs, 1H, H–NMe); 4.98–4.62 (m, 3H, H–
C(4’), H–C(5’, 5@)); 4.62–4.55 (m, 2H, OCH2); 3.44–3.31 (m, 3H, OCH2CH2, H–
C(2’)); 3.04 (d, 3H, HN–CH3); 2.48 (bs, 4H, CH3–C(6), H–C(2@)); 2.42 (s, 3H, tol–
CH3), 2.36 (s, 3H, tol–CH3).
Calc. For C37H36N6O9 (708.7): C, 62.70, H, 5.12, N, 11.86. Found: C, 62.36, H,
5.12, N, 11.54.
6-Methyl-2-(2-hydroxyethyl)amino-8-(2-deoxy-3,5-di-O-p-toluoyl- -D-ribofura-
nosyl)-O4-[2-(4-nitrophenyl)ethyl]-7(8H)-pteridinone (32). To a solution of 30 (2.0 g,
2.64 mmol) in CH2Cl2 (60 ml) was added ethanolamine (4.85 g, 80 mmol) and stirred at
r.t. for 6 h. It was neutralized with AcOH in CH2Cl2, evaporated and the residue purified
by CC (SiO2) with toluene/AcOEt 5:2. Evaporation of the product fraction and
coevaporation of the residue with CH2Cl2 yielded 1.36 g (70%) of a yellowish solid
1
foam. UV (MeOH): 203 (4.81), [215 (4.59)], 239 (4.70), [283 (4.23)], 348 (4.25). H-
NMR (CDCl3): 8.16 (d, 2H, o to NO2); 7.91 (d, 4H, arom. H); 7.47 (d, 2H, m to NO2);
7.31 (m, 1H, H–C(1’)); 7.23–7.12 (2d, 4H, o to CH3); 6.03 (m, 1H, H–C(3’)); 4.96 (bs,
1H, H–C(4’)); 4.72 (t, 2H, OCH2); 4.59 (m, 2H, H–C(5’,5@)); 3.88 (m, 2H, HN–
CH2CH2); 3.39 (m, 1H, H–C(2’)); 3.30 (t, 2H, OCH2CH2); 2.99 (bs, H, CH2CH2–OH);
2.48 (bs, 4H, CH3–C(6), H–C(2@)); 2.42 (s, 3H, tol–CH3), 2.37 (s, 3H, tol–CH3).
Calc. For C38H38N6O10 (738.8): C, 61.78, H, 5.18, N, 11.38. Found: C, 61.70, H,
5.26, N, 11.33.
6-Methyl-2-methylamino-8-(2-deoxy- -D-ribofuranosyl-O4-[2-(4-nitro-
phenyl)ethyl]-7(8H)-pteridinone (33). A solution of 31 (0.2 g, 0.28 mmol) in MeOH
(6 ml) and CH2Cl2 (4 ml) was treated with NaCN (65 mg) and stirring at r.t. for 24 h.
It was evaporated and then purified by CC with mixtures of CH2Cl2/MeOH 100:1
(85 ml), 50:1 (60 ml) and 10:1 (170 ml). The product fraction was evaporated and the
resulting foam recrystallized from toluene to give 82 mg (86%) yellowish crystals of
1
mp.> 300°C. UV (MeOH): 210 (4.57), 239 (4.29), 279 (4.19), 350 (4.24). H-NMR

ORDER
REPRINTS
64
Matysiak, Waldscheck, and Pfleiderer
(DMSO–d6): 8.19 (d, 2H, o to NO2); 7.64 (d, 3H, m to NO2, H–NMe); 7.19 (m, 1H,
H–C(1’)); 5.17 (m, 1H, HO); 4.83–4.51 (m, 3H, O–CH2, HO–C(3’)); 4.45 (m, 1H, H–
C(3’)); 3.88–3.61 (m, 2H, H–C(5’, 5@)); 3.55 (m, 1H, H–C(4’)); 3.27 (m, 2H,
OCH2CH2); 2.87 (m, 4H, HN–CH3, H–C(2’)); 2.27 (bs, 3H, CH3–C(6)); 2.01 (m, 1H,
H.C(2@)).
Calc. For C21H24N6O9 (472.5): C, 53.39, H, 5.12, N, 17.79. Found: C, 53.00, H,
5.17, N, 17.35.
6-Methyl-2-methylamino-8-(2-deoxy-3,5-di-O-p-toluoyl- -D-ribofuranosyl)-
4(3),7(8H)-pteridinedione (34). In a mixture of dioxane (4 ml) and acetonitrile (5 ml)
31 (0.2 g, 0.28 mmol) was dissolved and then treated with DBU (0.685 g, 4.5 mmol)
with stirring for 5 h. It was neutralized with AcOH in CH2Cl2, evaporated and the
residue put into a silica–gel column for chromatography with CH2Cl2 (80 ml) and
CH2Cl2/MeOH 40:1 (240 ml). The product fraction was evaporated, dried in high
vacuum to give 0.147 g (93%) of a yellowish solid of mp. 232–233°C (decomp.). UV
1
(MeOH): 202 (4.68), 217 (4.54), 237 (4.59), 296 (4.07), 347 (4.12). H-NMR (CDCl3):
11.69 (bs, 1H, H–N(3), 7.95 (m, 4H, arom. H); 7.46 (m, 2H, H–C(1’), H–NMe); 7.21
(2d, 4H, arom. H); 6.00 (m, H–C(3’)); 4.95–4.41 (m, 3H, H–C(4’), H–C(5’, 5@)); 3.34
(m, 1H, H–C(2’)); 3.07 (d, 3H, HN–CH3); 2.46 (s, 4H, CH3–C(6), H–C(2@)); 2.42 (s,
3H, CH3–tol); 2.37 (s, 3H, CH3–tol).
Calc. For C28H29N5O7 ꢀ 2 H2O (571.5): C, 56.74, H, 5.82, N, 12.25. Found: C,
56.73, H, 5.42, N, 12.05.
6-Methyl-2-(2-hydroxyethyl)amino-8-(2-deoxy-3,5-di-O-p-toluoyl- -D-ribofura-
nosyl)-4(3),7(8H)-pteridinedione (35). Analogous to the preceding procedure with
32 (0.411 g, 0.56 mmol) in dioxane (5 ml) and acetonitrile (15 ml) with DBU (1.52 g,
10 mmol). Purification by CC with CH2Cl2 (200 ml), CH2Cl2/MeOH 19:1 (200 ml)
and CH2Cl2/MeOH 9:1 (200 ml) to give after evaporation and recrystallization from
MeOH 0.233 g (71%) of yellowish crystals of mp. 207–208°C. UV (MeOH): 202
1
(4.77), 222 (4.61), 237 (4.70), 297 (4.14), 345 (4.21). H-NMR (CDCl3): 11.02 (bs,
1H, H–N(3), 7.90 (m, 4H, arom. H); 7.35 (m, 5H, arom. H, H–C(1’)); 7.05 (bs, 1H,
H–NCH2CH2); 5.90 (m, H–C(3’)); 4.95 (bs, 1H, CH2CH2–OH); 4.67–4.45 (m, 3H,
H–C(4’), H–C(5’, 5@)); 3.58–3.35 (m, 4H, CH2CH2); 3.15 (m, 1H, H–C(2’)); 3.07
(d, 3H, HN–CH3); 2.38 (s, 4H, CH3–C(6), H–C(2@)); 232 (s, 3H, CH3–tol); 2.27
(s, 3H, CH3–tol).
Calc. For C30H31N5O8 (589.6): C, 61.11, H, 5.30, N, 11.88. Found: C, 61.11, H,
5.42, N, 11.67.
6-Methyl-2-{2-[2-(4-nitrophenyl)ethoxycarbonyloxy]ethyl}amino-8-(2-deoxy-
3,5-di-O-p-toluoyl- -D-ribofuranosyl)-O4-[2-(4-nitrophenyl)ethyl]-7(8H)-pteridinone
(36). Compound 32 (0.25 g, 0.34 mmol) was coevaporated with dry pyridine
(3 ꢀ 10 ml) and then dissolved in dry pyridine (4 ml). Under exclusion of moisture 2-
(4-nitrophenyl)ethoxy-carbonyl chloride (85 mg, 0.37 mmol) was added and then
stirred at 40°C for 30 h. It was evaporated, coevaporated with toluene, the residue
dissolved in little CH2Cl2 and put on a silica–gel column for chromatography with
toluene/AcOEt 7:2. The product fraction was evaporated and coevaporated several
times with CH2Cl2 to give after drying in high vacuum 0.28 g (88%) of a colorless

ORDER
REPRINTS
Nucleosides. LXV
65
solid foam. UV (MeOH): 202 (4.88), 239 (4.73), [270 (4.4)], 346 (4.26). 1H-NMR
(CDCl3): 8.16 (m, 4H, o to NO2); 7.92 (d, 4H, arom. H); 7.48 (d, 2H, m to NO2); 7.37
(d, 2H, m to NO2); 7.20 (m, 5H, H–C(1’), o to CH3); 6.01 (m, 2H, H–N, H–C(3’));
4.94 (bs, 1H, H–C(4’)); 4.74 (t, 2H, OCH2); 4.58 (m, 2H, H–C(5’,5@)); 4.35 (m. 4H,
OCOOCH2CH2, HNCH2CH2OCO); 3.76 (m, 2H, OCOOCH2CH2); 3.38 (m, 1H, H–
C(2’)); 3.30 (t, 2H, OCH2CH2); 3.06 (bs, H, HNCH2CH2OCO); 2.50 (bs, 4H, CH3–
C(6), H–C(2@)); 2.44 (s, 3H, tol–CH3), 2.38 (s, 3H, tol–CH3).
Calc. For C47H45N7O14 (931.9): C, 60.58, H, 4.87, N, 10.52. Found: C, 60.78, H,
5.01, N, 10.19.
REFERENCES
1. Mu¨nch, U.; Pfleiderer, W. Nucleosides LXIV. Base-labile protecting groups for
oligoribonucleotide synthesis. Helv. Chim. Acta 2002, 85, 1504–1517.
2. Schmid, H.; Schranner, M.; Pfleiderer, W. Nucleoside XI. Synthese von
Isoxanthopterin-N-8-b-D-ribofuranosid—ein strukturanaloges Nucleosid des Gua-
nosins. Chem. Ber. 1973, 106, 1952–1975.
3. Ritzmann, G.; Ienaga, K.; Pfleiderer, W. Nucleoside XXIV. Verbesserte Synthesen
von Lumazinnucleosiden. Liebigs Ann. Chem. 1977, 1217–1234.
4. Harris, R.; Pfleiderer, W. Nucleoside XXXVI. Synthese und Eigenschaften des 4-
amino-8-b-D-ribofuranosyl-7(8H)-pteridones sowie seiner 2- und 6-Phenylderivate.
Liebigs Ann. Chem. 1981, 1457–1468.
5. Hawkins, M.E.; Pfleiderer, W.; Mazumder, A.; Pommier, Y.G.; Balis, F.M.
Incorporation of a fluorescent guanosine analog into oligonucleotides and its
application to a real time assay for the HIV-1 integrase 3’-processing reaction.
Nucleic Acids Res. 1995, 23, 2872–2880.
6. Jungmann, O.; Pfleiderer, W. A new efficient method in nucleoside synthesis.
Tetrahedron Lett. 1996, 37, 8355–8358.
7. Melguizo, M.; Gottlieb, M.; Charubala, R.; Pfleiderer, W. Nucleosides LXII.
Synthesis of 6-methyl-8-(2-deoxy-b-D-ribofuranosyl)-isoxanthopterin and deriva-
tives. Nucleosides Nucleotides 1998, 17, 175–186.
8. Lehbauer, J.; Pfleiderer, W. Nucleotides LXIX. Synthesis of phosphoramidite
building blocks of isoxanthopterin N8-(2’-deoxy-b-D-ribonucleosides): new fluo-
rescence markers for oligonucleotide synthesis. Helv. Chim. Acta 2001, 84, 2330–
2342.
9. Hawkins, M.E.; Pfleiderer, W.; Balis, F.M.; Porter, D.; Knutson, J.R. Fluorescence
properties of pteridine nucleoside analogs as monomers and incorporated into
oligonucleotides. Anal. Biochem. 1997, 244, 86–95.
10. Bannwarth, W.; Mu¨ller, F. Energy transfer from a lumazine (= Pteridine-2, 4-
(1H, 3H)dione) chromophore to bathophenanthroline-ruthenium (II) complexes
during hybridization processes of DNA. Helv. Chim. Acta 1991, 74, 2000–
2008.
11. Bannwarth, W.; Pfleiderer, W.; Mu¨ller, F. Energy transfer within oligonucleotides
from a lumazine chromophore to bathophenanthroline–ruthenium(II) complexes.
Helv. Chim. Acta 1991, 74, 1991–1999.
12. Driscoll, S.; Hawkins, M.E.; Balis, F.M.; Pfleiderer, W.; Laws, W.R. Fluorescence

ORDER
REPRINTS
66
Matysiak, Waldscheck, and Pfleiderer
properties of a new guanosine analog incorporated into small oligonucleotides.
Biophys. J. 1997, 73, 3277–3286.
13. Birkofer, L.; Ritter, A.; Ku¨hlthau, H.P. Alkylierungen und Glykosidierungen u¨ber
Silyl-Derivative. Chem. Ber. 1964, 97, 934–945.
14. Birkofer, L.; Ritter, A. Die Silylierung als Hilfsmittel in der Organischen Synthese.
Angew. Chem. 1965, 77, 414–426.
15. Kiriasis, L.; Pfleiderer Nucleoside XLV. Synthese von 8-b-D-ribofuranosyl-
leukopterin. Nucleosides Nucleotides 1989, 8, 1345–1358.
¨
16. Ritzmann, G.; Ienaga, K.; Kiriasis, L.; Pfleiderer, W. Nucleoside XXXIII. Uber die
Synthese des 7-Oxo-8-b-D-ribofuranosyl-7,8-dihydrolumazins und seines 6-Methyl-
derivates. Chem. Ber. 1980, 113, 1535–1548.
17. Johns, C.O.; Baumann, E.J. Researches in purines. On 2-methylmercapto-6,8-
dioxypurine and 2-methylmercapto-6-oxy-8-aminopurine. J. Biol. Chem. 1913, 14,
381–388.
Received August 6, 2003
Accepted September 18, 2003

Request Permission or Order Reprints Instantly!
Interested in copying and sharing this article? In most cases, U.S. Copyright
Law requires that you get permission from the article’s rightsholder before
using copyrighted content.
All information and materials found in this article, including but not limited
to text, trademarks, patents, logos, graphics and images (the "Materials"), are
the copyrighted works and other forms of intellectual property of Marcel
Dekker, Inc., or its licensors. All rights not expressly granted are reserved.
Get permission to lawfully reproduce and distribute the Materials or order
reprints quickly and painlessly. Simply click on the "Request Permission/
Order Reprints" link below and follow the instructions. Visit the
U.S. Copyright Office for information on Fair Use limitations of U.S.
copyright law. Please refer to The Association of American Publishers’
Fair Use in the Classroom.
(AAP) website for guidelines on
The Materials are for your personal use only and cannot be reformatted,
reposted, resold or distributed by electronic means or otherwise without
permission from Marcel Dekker, Inc. Marcel Dekker, Inc. grants you the
limited right to display the Materials only on your personal computer or
personal wireless device, and to copy and download single copies of such
Materials provided that any copyright, trademark or other notice appearing
on such Materials is also retained by, displayed, copied or downloaded as
part of the Materials and is not removed or obscured, and provided you do
not edit, modify, alter or enhance the Materials. Please refer to our
Website
User Agreement for more details.
Request Permission/Order Reprints
Reprints of this article can also be ordered at
http://www.dekker.com/servlet/product/DOI/101081NCN120027817