Preparation and antiviral properties of new acyclic, achiral nucleoside analogues: 1- or 9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]nucleobases and 1- or 9-[2,3-dihydroxy-2-(hydroxymethyl)propyl]nucleobases.

Article abstract of DOI:10.1039/b316304k

Acyclic, achiral nucleoside derivatives 1b-e of adenine, cytosine, 5-methylcytosine, and guanine, containing a 3-hydroxy-2-(hydroxymethyl)prop-1-enyl group on N-1 or N-9, have been prepared analogously to the previously described thymine derivative 1a. In contrast to the adenine and guanine derivatives, the cytosine derivative 9 was unstable, and was obtained in a low yield due to side reactions. These include cleavage of the propenyl group from the base, and the formation of a bicyclic compound. The thymine derivative, although stable under neutral conditions, likewise underwent a reversible cyclization reaction (Michael addition) in the presence of acids or bases. The 5-methylcytosine derivative was stable under neutral and basic conditions. Four other nucleoside derivatives 26a-d containing a 2,3-dihydroxy-2-(hydroxymethyl)propyl group on N-1 or N-9, three of which are new, have likewise been prepared. All compounds were evaluated as antiviral agents against HIV-1 and HSV-1 but were devoid of antiviral activity.

Full text of DOI:10.1039/b316304k

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Preparation and antiviral properties of new acyclic, achiral
nucleoside analogues: 1- or 9-[3-hydroxy-2-(hydroxymethyl)prop-1-
enyl]nucleobases and 1- or 9-[2,3-dihydroxy-2-(hydroxymethyl)-
propyl]nucleobases
Thomas Boesen,a Christian Madsen,a Daniel Sejer Pedersen,a Brian M. Nielsen,a
Asger B. Petersen,a Michael Å. Petersen,a Michael Munck,a Ulla Henriksen,a Claus Nielsenb
and Otto Dahl*a
a Department of Chemistry, University of Copenhagen, The H. C. Ørsted Institute,
Universitetsparken 5, DK-2100 Copenhagen, Denmark.
E-mail: dahlo@kiku.dk; Fax: ϩ45 35320212; Tel: ϩ45 35320176
b Retrovirus Laboratory, Department of Virology, Statens Serum Institut,
DK-2300 Copenhagen, Denmark
Received 16th December 2003, Accepted 23rd February 2004
First published as an Advance Article on the web 16th March 2004
Acyclic, achiral nucleoside derivatives 1b–e of adenine, cytosine, 5-methylcytosine, and guanine, containing a
3-hydroxy-2-(hydroxymethyl)prop-1-enyl group on N-1 or N-9, have been prepared analogously to the previously
described thymine derivative 1a. In contrast to the adenine and guanine derivatives, the cytosine derivative 9 was
unstable, and was obtained in a low yield due to side reactions. These include cleavage of the propenyl group from
the base, and the formation of a bicyclic compound. The thymine derivative, although stable under neutral
conditions, likewise underwent a reversible cyclization reaction (Michael addition) in the presence of acids or bases.
The 5-methylcytosine derivative was stable under neutral and basic conditions. Four other nucleoside derivatives
26a–d containing a 2,3-dihydroxy-2-(hydroxymethyl)propyl group on N-1 or N-9, three of which are new, have
likewise been prepared. All compounds were evaluated as antiviral agents against HIV-1 and HSV-1 but were
devoid of antiviral activity.
Introduction
Nucleoside analogues, as well as modified oligonucleotides,
have been widely used in recent years to regulate viral growth
and cellular gene expression.1 A host of analogues have been
prepared and evaluated in order to obtain compounds with
improved stability towards enzymatic cleavage, better cell
membrane penetration, and higher and more selective binding
to their substrates, when compared with their natural counter-
Fig. 1
parts. In the antiviral field, successful nucleoside analogues have
often been found accidentally, since it is difficult to design
investigate the potential of this type of nucleoside analogue in
compounds that are sufficiently selective towards viral enzymes.
Although many are close analogues of nucleosides, e.g. the HIV
drug 3Ј-azido-2Ј,3Јdideoxythymidine (AZT),2 others are acyclic
analogues, e.g. the herpes virus drug acyclovir.3 In the antisense
and antigene field, downregulation of gene expression follows
more recognized patterns, such as established rules for efficient
Watson–Crick binding and base stacking, a good knowledge of
the factors which lead to nuclease stability, and the importance
of preorganization. In the antisense field, the first successfully
modified oligonucleotides were conservatively modified com-
pounds like phosphorothioates and methylphosphonates,
but in recent years many highly modified oligonucleotides with
improved binding properties have been developed, e.g. PNA,4
LNA,5 and anhydrohexitol oligonucleotides.6
Recently we prepared a new acyclic, achiral nucleoside
analogue, 1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]thymine
(Fig. 1, 1a).7 This analogue is quite different from a normal
thymine nucleoside, but molecular models and geometry calcu-
lations indicate that it could be a good nucleoside mimic.8 How-
ever, preliminary binding studies of oligonucleotides containing
one or two molecules of 1a towards complementary DNA and
RNA strands showed a reduced binding (∆T m Ϫ2 to Ϫ6.5 ЊC
per single introduced nucleotide modification).9 In order to
more detail we decided to prepare and study four other nucleo-
base analogues. This paper describes an improved synthesis of
1a, the synthesis of the cytosine analogue 1b, the 5-methyl-
cytosine analogue 1c, the adenine analogue 1d, the guanine
analogue 1e, the synthesis of some related trihydroxyalkyl-
nucleosides 26a–d, and some of their antiviral properties.
Results and discussion
Preparation of 1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]-
pyrimidines
The previously described epoxide route to the thymine deriv-
ative 1a7 is shown in Scheme 1. This route gave 1a in a reason-
able yield, but the elimination step was found to give highly
variable yields of 5a depending on the quality of the base,
potassium tert-butoxide. When potassium tert-butoxide was
very dry, the yield of 5a was approximately 40% after purifi-
cation. However, samples of potassium tert-butoxide that had
absorbed water gave no 5a but only the isomeric alkenes 6.
Contrary to our first report,7 6 could not be isomerized to 5a
by treatment with strong bases. Better results have now been
obtained using sodium tert-amyloxide, freshly prepared from
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
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
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and benzamide were isolated from of the CHCl3 phase by
chromatographic separation.
The structure of 7 was ascertained by 1H NMR and MS, and
1
by its slow hydrolysis to CBz and presumably 8.11 Thus the H
NMR spectrum (DMSO-d6) of 7 showed two vinylic proton
signals, one primary alcohol, and one methoxy group. Since
the compound is an aminal it should hydrolyze easily to the
aldehyde 8 and CBz as was observed. The formation of 7 from
5b and BCl3 can be rationalized as follows (Scheme 3). Removal
of one benzyl group from 5b gives an intermediate that can
react in two different ways, to give either the cyclic boron ester
which leads to 9 or a stabilized carbocation which leads to 7
after quenching with MeOH. Apparently the reaction proceeds
mainly via the stabilized carbocation at Ϫ78 ЊC, whereas at 0 ЊC
a substantial part of the reaction occurs via the cyclic ester to
give 9. However, the N-4 unprotected analogue of 5b reacted
with BCl3 at 0 ЊC solely via the cyclic ester to give 1b and 10
(data not shown).
Scheme 1
anhydrous tert-amyl alcohol and NaH in dry toluene, instead
of potassium tert-butoxide. This base gave 5a reproducibly in a
50–55% yield after purification. The last step, removal of the
benzyl groups, was also improved. 1a Was found to be some-
what unstable in the strongly acidic solution produced by
methanolysis of excess boron trichloride. However, it was
obtained in a reproducible yield of ca. 65% after purification
when the reaction mixture was concentrated and neutralized
quickly, as described in the Experimental section.
A similar synthesis of the cytosine derivative 1b was
attempted from N-4-benzoylcytosine10 (CBz, Scheme 2). The
preparation of 3b, 4b, and 5b proceeded just as well as for
the analogous thymine derivatives. However, when the thymine
procedure (6 mol eq. of BCl3 in CH2Cl2, 4 h at Ϫ78 ЊC) to
convert 5b to 9 was attempted, only CBz was isolated from a
mixture of products that only contained small amounts of 9.
Under modified conditions (10 mol eq. of BCl3 in CH2Cl2, 1 h
at 0 ЊC), 9 could be obtained in a yield of approximately 35%
together with a mixture of several other compounds. The
crude mixture was partitioned between CHCl3 and H2O, and 9
and an unexpected compound 10 (see below for the structure
elucidation of 10) were isolated from the aq. phase as a mixture
(3 : 1 according to NMR), from which only 10 could be crystal-
lized in a pure state. However, after conversion to the 4,4Ј-
dimethoxytrityl (DMT) derivatives 11 and 12 the mixture
could be separated by chromatography, and deprotection of
11 produced 1b as the hydrochloride. Of the other products 7
Scheme 3
1
The structure of 10 was determined by H NMR, MS, and
elemental analysis. Characteristic NMR signals are one OH
signal only, the ABX system for the 5- and 6-protons of the
perhydropyrimidine ring, the AB system for the CH2 group in
the other ring, and the multiplet signal for the CH2OH. The
absence of aromatic signals and the presence of only one NH
signal shows that the benzamide group has been hydrolysed
to an oxo group, i.e., that the compound is a reduced uracil
derivative. The bicyclic system is undoubtedly formed by a
Michael addition of one of the OH groups of 9 to the 5,6-
double bond of cytosine; whether the 4-NHBz group is
removed before or after the ring closure is not known.
The Michael addition leading to bicyclic compounds like 10
to our knowledge has no close equivalents in the literature, but
conjugate addition of good nucleophiles to the 5,6-double bond
of cytosine or uracil derivatives is well-known.12 Compounds 1a
and 1b both undergo a similar Michael addition reaction to give
14 and 15, respectively (Scheme 4). The rate of cyclization,
however, is very different for 1a and 1bؒHCl.
Scheme 4
Thus, 1a is very stable in solution under neutral conditions,
but both acids and bases catalyze the formation of 14. Triethyl-
amine, but not pyridine, gave, after a few hours in MeOH
at rt, an equilibrium mixture of 1a and 14 from which 14 could
be precipitated and characterized. p-Toluenesulfonic acid in
MeOH likewise catalyzed the reaction, although more slowly,
and the reaction was less clean. In contrast 1b (as its hydro-
chloride) is unstable in solution and was found to partly cyclize
to 15 (and/or the corresponding uracil derivative 10) during an
overnight recording of its 13C NMR spectrum in CD3OD.
Apparently the 5-CH3 group of thymine protects the double
Scheme 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
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bond from the Michael addition of weak nucleophiles suffi-
ciently to make 1a stable under neutral or near neutral
conditions. Based on these observations we decided to prepare
the 5-methylcytosine (MeC) derivative 1c.
The 5-methylcytosine derivative 1c was prepared in two steps
from the thymine derivative 5a (Scheme 5). The 4-oxo group
of 5a was transformed to an amino group using standard
methods13 to give 5c, which could be debenzylated with BCl3
to give 1c in 70% yield. Unlike 1a and 1b.HCl, 1c was stable in
solution under both neutral and basic conditions. However, the
N-4-benzoyl derivative of 1c was prone to cyclization (data not
shown).
Scheme 5
Scheme 7
At present we do not fully understand the factors which
govern the tendency of 1a–c and their derivatives to cyclize.
A more thorough investigation of this interesting reaction is
under way.
ceeded well with BCl3, however, the product was difficult to
purify due to its high polarity. Crude 21 was therefore trans-
formed to the bis-dimethoxytrityl derivative 22 which after
removal of the isobutyryl group gave 23. From 23 the
unprotected 1e could be isolated by precipitation from a
CH2Cl2 solution upon addition of CCl3COOH and Et3SiH.15 1e
Has previously been described in a Japanese patent without
characterization, apart from 1H NMR.16
Preparation of 9-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]-
purines
The procedure developed for thymine worked well for adenine
(Scheme 6). When 2 was treated with adenine the main product
was 17 together with the N-7 isomer that was easily removed by
column chromatography. Purified 17 was protected on N-6 with
a 4-tert-benzoyl group to give 3d; this protection group gave
nucleosides that were more soluble than when a benzoyl group
was employed. The remaining steps proceeded satisfactorily to
give a mixture of 18 and the N-6 unprotected product 1d that
were separated by column chromatography.
Preparation of 1- or 9-[2,3-dihydroxy-2-(hydroxymethyl)propyl]-
nucleobases
The trihydroxyalkyl nucleobase derivatives 26a–d (Scheme 8)
could be prepared from the dibenzyl protected epoxide 2, but a
simpler route was developed from the dibenzoyl protected
epoxide 24 (Scheme 8). The epoxide 24 has been reported as
a by-product previously without proper characterization.17
We prepared it in two steps starting from 3-chloro-2-(chloro-
methyl)propene in an overall yield of 70%. The reactions of 24
with an excess of thymine, N-4-benzoylcytosine, N-6-benzoyl-
adenine, or 2-amino-6-chloropurine and a base predominantly
gave the N-1 or N-9 substituted products 25a–d together
with small amounts (0–10%) of regioisomers or dialkylated
products. The alkylations to give 25a–b were shown to have
occurred at N-1 by NOE, and 25c–d were shown to be the N-9
1
alkylated products by comparison of their 13C and H NMR
data with those of known N-9 alkylated compounds.18,19 Treat-
ment with ammonia removed the benzoyl groups of 25a–c to
give 26a–c, and 25d was converted to 26d with hydrochloric
acid. The adenine compound 26c has been prepared previously
by another route.20
Scheme 6
The guanine derivative 1e was prepared from 2 and 2-amino-
6-chloropurine (Scheme 7). This reaction gave 19 without
discernible amounts of the N-7 isomer, whereas N-2-isobutyryl-
guanine and 2 gave a mixture of the N-7 and the N-9 isomer
with the former dominating. 19 was transformed to the guanine
derivative 20 with 3-hydroxypropionitrile and NaH14 and the
product protected at N-2 with isobutyric anhydride to give 3e.
Phosphitylation and oxidation with sulfur to 4e proceeded as
usual, but the elimination to 5e was at first troublesome. When
4e was dried by coevaporation with pyridine, traces of pyridine
gave rise to a mixture of 5e and the regioisomeric alkenes
(analogous to 6, Scheme 1). Since these alkenes were difficult
to separate it was important to obtain a highly regioselective
elimination. Fortunately it was possible to remove traces of
pyridine from 4e by co-evaporation with toluene and thereby
obtain pure 5e. Removal of the benzyl groups to give 21 pro-
Scheme 8
Antiviral activity
Compounds 1a, 1c–e, and 26a–d were tested against HIV-1 in
MT4 cell cultures infected with wildtype HIV-1 (strain IIIB),
and against HSV-1 in vero cells, as described earlier.21,22 All
compounds were devoid of any activity at 100 µM and showed
little or no cytotoxicity. The lack of activity might be due to the
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
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relatively short distance between the hydroxy groups and the
nucleobases in 1 and 26. Thus analogues with one additional
carbon atom are active against a variety of viruses, e.g., penci-
clovir 27 (Fig. 2) is active against HSV-1, HSV-2, and VZV,1a
9-[4-hydroxy-3-(hydroxymethyl)-2-butenyl]guanine 28 (but
not the adenine analogue) is active against HSV-2,23 and R-
Adenallene 29 and R-Cytallene 30 (but not the guanine or
thymine analogues) are active against HIV-1.1a However, the
closely related 31 is devoid of antiviral activity.24
CH2Cl2–EtOAc 3 : 2 v/v, followed by crystallisation from
EtOAc–heptane, gave 5a (1.66 g, 53%) as colourless crystals,
mp 92–94 ЊC (lit.7 mp 92–94 ЊC).
1-[3-Hydroxy-2-(hydroxymethyl)prop-1-enyl]thymine 1a
To a stirred solution of 5a (1.18 g, 3.0 mmol) in dry CH2Cl2
(90 ml) at Ϫ78 ЊC under N2 was added dropwise BCl3 (1 M in
CH2Cl2, 18.0 ml, 18 mmol) during 5 min. Stirring was con-
tinued at Ϫ78 ЊC for 4 h, followed by dropwise addition of
MeOH–CH2Cl2 (1 : 1 v/v, 25 ml) at Ϫ78 ЊC. The cooling bath
was removed and the solvents quickly removed in vacuo to give
a residue that was immediately dissolved in MeOH–CH2Cl2
(1 : 2 v/v, 45 ml), and solid NaHCO3 (approx. 200 mg) was
added with stirring in small portions until the solution was
neutral on wet pH paper. The solids were removed by filtration
and the filtrate concentrated in vacuo to give a brown residue
that was dissolved in a mixture of hexane (40 ml) and water
(15 ml). The aqueous phase was extracted with hexane (2 ×
30 ml) followed by concentration of the aqueous phase in
vacuo. The residue was purified by normal gravity column
chromatography, eluted with EtOAc–MeOH 9 : 1 v/v, to give
pure 1a (0.41 g, 64%) as colourless crystals, mp 166–168 ЊC
(lit.7 mp 166–169 ЊC).
Fig. 2
Conclusion
An epoxide route, previously developed for the thymine DNA
analogue 1a, has been shown to be useful for the preparation
of similar DNA analogues 1d and 1e derived from adenine
and guanine. In the case of cytosine the last synthetic step was
low yielding due to several side reactions, and the analogue 1b
as the hydrochloride was unstable in solution. However, the
5-methylcytosine derivative 1c could be made from the thymine
derivative 5a and was found to be stable. An unexpected pro-
pensity of the pyrimidine derivatives to undergo an intra-
molecular Michael addition to bicyclic compounds 10 and
14–16 was discovered. The compounds 1a, 1c–1e, and some
related saturated derivatives 26a–d, were evaluated as antiviral
agents against HIV-1 and HSV-1, but were found to be without
activity at 100 µM. Work is in progress to build 1c–1e into
oligodeoxyribonucleotides in order to evaluate their potential
as modifying units in antisense oligonucleotides.
N-4-Benzoyl-1-[3-benzyloxy-2-(benzyloxymethyl)-2-hydroxy-
propyl]cytosine 3b
NaH (60% in oil, 3.6 g, 90 mmol) was added under N2 to a
suspension of N-4-benzoylcytosine (19.4 g, 90 mmol) in dry
DMF (600 ml) and the mixture stirred for 2 h at rt. Then 2 (12.8
g, 45 mmol) was added and the mixture heated to 110 ЊC for
24 h. After cooling sat. aq. NH4Cl (250 ml) and H2O (100 ml)
were added and the mixture stirred for 15 min. The precipitate
was removed by filtration and washed with CH2Cl2 (6 ×
100 ml). The filtrate was extracted with EtOAc (650 ml ϩ
4 × 250 ml) and the combined organic phases wre dried
(Na2SO4) followed by evaporation in vacuo. The residue was
suspended in EtOAc (300 ml) and filtered through silica gel.
The silica gel was washed extensively with EtOAc and the
filtrate evaporated in vacuo to pale yellow crystals that were
recrystallized from EtOAc–heptane to give pure 3b (15.4 g,
68%) as colourless crystals, mp 143–144.5 ЊC. NMR (CDCl3):
δH 9.1 (1H, br s, NH), 7.94 (2H, d, J 7, Ar),7.71 (1H, d, J 7,
H-6), 7.60 (1H, t, J 7, Ar), 7.50 (2H, d, J 7, Ar), 7.40–7.25 (11H,
m, Ar ϩ H-5), 4.51 (4H, s, PhCH2), 4.45 (1H, s, OH), 4.20 (2H,
s, NCH2), 3.51 (4H, AB system, ∆ 14.7 Hz, JAB 9.7, BnOCH2).
δC 166.5, 162.1, 157.0, 150.7, 137.4, 132.9, 132.7, 128.7, 128.1,
128.0, 127.5, 127.4, 96.3, 74.3, 73.3, 71.9, 54.0. FABϩMS: 500.2
(M ϩ Hϩ calc. 500.2) (Found: C, 69.7; H, 5.95; N, 8.5. Calc. for
C29H29N3O5: C, 69.7; H, 5.85; N, 8.4%).
Experimental
2,2-Bis(benzyloxymethyl)oxirane (2),7 1-[3-benzyloxy-2-(benz-
yloxymethyl)-2-hydroxypropyl]thymine (3a),7 1-[3-benzyloxy-
2-(benzyloxymethyl)-2-(diethoxythiophosphoryloxy)propyl]-
thymine (4a),7 and N-4-benzoylcytosine,10 were prepared
according to literature procedures. Other chemicals were 97–
99% pure from Aldrich, Fluka, Sigma, or Merck, unless other-
wise stated. Solvents were HPLC grade from LABSCAN, of
which CH2Cl2, DMF, pyridine, tert-amyl alcohol and toluene
were dried over molecular sieves (4 Å from Grace Davison)
and THF freshly distilled from Na-benzophenone to a water
content below 20 ppm, measured on a Metrohm 652 KF-
coulometer. TLC was run on Merck 5554 silica 60 aluminium
sheets, LC on either Merck 9385 silica 60 (0.040–0.063 mm) for
normal gravity and flash chromatography, or Merck 15111
silica 60 (0.015–0.040 mm) for dry column vacuum chromato-
graphy.25 NMR spectra (reference tetramethylsilane for δH and
δC, external 85% H3PO4 for δP, J values are given in Hz) were
run on a Varian Mercury 300 MHz spectrometer, and FAB MS
data obtained on a JEOL HX 110/110 mass spectrometer with
m-NBA as the matrix.
N-4-Benzoyl-1-[3-benzyloxy-2-(benzyloxymethyl)-2-(diethoxy-
thiophosphoryloxy)propyl]cytosine 4b
To a stirred solution of 3b (5.00 g, 10.0 mmol) in dry pyridine
(50 ml) under N2 at rt was added dropwise diethyl phosphoro-
chloridite (1.65 ml, 11.5 mmol). After 45 min S8 (0.40 g, 12.5
mmol S) was added, and the mixture was stirred for 1.5 h,
followed by evaporation in vacuo. The residue was dissolved
in CH2Cl2 (250 ml) and the solution extracted with sat. aq.
NaHCO3 (2 × 100 ml), brine (100 ml), dried (MgSO4), and
evaporated in vacuo. The residue was crystallized from EtOAc–
hexane to give 4b (4.05 g, 62%) as colourless crystals, mp 102–
103 ЊC. NMR (CDCl3): δH 8.5 (1H, br s, NH), 8.0 (3H, m, Ar ϩ
H-6), 7.65 (1H, t, J 7, Ar), 7.55 (2H, d, J 7, Ar), 7.35–7.20 (11H,
m, Ar ϩ H-5), 4.51 (2H, s, NCH2), 4.49 (4H, AB system, ∆ 20.1
Hz, JAB 11.4, PhCH2), 4.14–4.02 (6H, m, Et ϩ BnOCHA), 3.82
(2H, d, JAB 10.3, BnOCHB), 1.25 (6H, t, J 6.9, Et). δC 161.9,
150.2 br, 137.6, 133.1, 129.0 128.2, 127.8, 127.7, 127.5, 86.2 (d,
JPC 9), 73.5, 69.9, 64.5 (d, JPC 6), 51.8, 15.8 (d, JPC 8). δP 59.2.
1-[3-Benzyloxy-2-(benzyloxymethyl)prop-1-enyl]thymine 5a
To dry tert-amyl alcohol (3.50 g, 40 mmol) in dry toluene (40
ml) was added NaH (60% in oil, 1.60 g, 40 mmol) and the
mixture was stirred at rt for 1 h. A solution of 1-[3-benzyloxy-
2-(benzyloxymethyl)-2-(diethoxythiophosphoryloxy)propyl]-
thymine (4a) (4.50 g, 8.0 mmol, dried by co-evaporation with
dry toluene) in dry toluene (40 ml) was added, and the mixture
stirred at rt for 3 days. The mixture was quenched with 4
M aq. HCl (20 ml) and the solvents removed in vacuo.
Purification by flash column chromatography, eluted with
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
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FABϩ MS: 652.5 (M ϩ Hϩ calc. 652.2)(Found: C, 60.6; H, 5.9;
N 6.4; S, 4.8. Calc. for C33H38N3O7PS: C, 60.8; H, 5.9; N, 6.45;
S, 4.9%).
N-4-Benzoyl-1-[3-dimethoxytrityloxy-2-(dimethoxytrityloxy-
methyl)prop-1-enyl]cytosine 11 and 3-(dimethoxytrityloxy-
methyl)-6,8-dioxo-7,8,9,9a-tetrahydro-2H,6H-pyrimido[6,1-b]-
[1.3]oxazine 12
N-4-Benzoyl-1-[3-benzyloxy-2-(benzyloxymethyl)prop-1-enyl]-
cytosine 5b
To a solution of 9 and 10 (0.087 g, ca. 3 : 1) in dry pyridine
(2 ml) under N2 was added dimethoxytrityl chloride (0.39 g,
1.15 mmol), and the mixture was stirred for 1.5 h at rt followed
by concentration in vacuo. The residue was partitioned between
CH2Cl2 (10 ml) and H2O (10 ml), and the organic phase was
extracted with sat. aq. NaHCO3 (3 × 5 ml), brine (5 ml), dried
(MgSO4) and the solvent removed in vacuo. Purification of the
residue by normal gravity column chromatography, eluted with
EtOAc–hexane 3 : 2 v/v, gave pure 12 (0.020 g, 0.04 mmol,
14%), Rf 0.39 (EtOAc–hexane 3 : 2 v/v), then pure 11 (0.120 g,
0.13 mmol, 46%) as colourless crystals, Rf 0.22 (EtOAc–hexane
3 : 2 v/v). 11: NMR (CDCl3): δH 8.6 (1H, br, NH), 7.90 (2H, d,
J 7, Ar), 7.65–7.15 (23H, m, Ar ϩ H-5 ϩ H-6), 7.04 (1H, br,
To a stirred solution of dry tert-amyl alcohol (0.80 ml, 6 mmol)
in dry toluene (30 ml) under N2 at rt was added NaH (60%
in oil, 0.24 g, 6.0 mmol). After stirring for 1 h 4b (0.78 g,
1.2 mmol) was added, and the stirring was continued for 4 h at
rt, followed by neutralization with 2 M aq. HCl at 0 ЊC. CHCl3
(90 ml) was added, and the mixture washed with sat. aq.
NaHCO3 (3 × 50 ml) and brine (50 ml). The organic phase was
dried (MgSO4), and the solvent removed in vacuo to give light
yellow crystals. Purification by flash column chromatography,
eluted with CH2Cl2–MeOH 97 : 3 v/v, gave 5b (0.302 g, 52%)
as colourless crystals, mp 153–154 ЊC. NMR (CDCl3): δH 8.9
(1H, br, NH), 7.93 (2H, d, J 7, Ar), 7.78 (1H, d, J 7.3, H-6), 7.60
(1H, t, J 7, Ar), 7.50 (2H, d, J 7, Ar), 7.45–7.25 (11H, m, Ar ϩ
N–CH᎐C), 6.85 (4H, d, J 8.8, Ar), 6.75 (4H, d, J 8.8, Ar), 3.93
᎐
(2H, d, J 1.2, CH C᎐CH), 3.79 and 3.73 (2 × 6H, 2 × s, OCH ),
᎐
᎐
2
3
H-5), 6.94 (1H, br t, N–CH᎐C), 4.57 and 4.49 (2 × 2H, 2 × s,
3.59 (2H, s, CH C᎐CH). FABϩ MS: 906.6 (M ϩ Hϩ calc.
᎐
2
PhCH2), 4.19 (2H, d, J 1.2, BnOCH2), 4.02 (2H, s, BnOCH2).
FABϩ MS: 482.1 (M ϩ Hϩ calc. 482.2) (Found: C, 71.7; H, 5.6;
N, 8.7. Calc. for C29H27N3O4: C, 72.3; H, 5.65; N, 8.7%).
906.4). 12: NMR (CDCl3): δH 7.84 (1H, br, NH), 7.4–7.2 (9H,
m, Ar), 7.07 (1H, br s, N–CH᎐C), 6.84 (4H, d, J 9.1, Ar), 5.05
᎐
(1H, X of ABX system, JAX 5.3, JBX 10.3, O–CH–N), 4.35 (2H,
br AB system, ∆ 19.3 Hz, JAB 16.0, OCH2 (ring)), 3.80 (6H, s,
OCH3), 3.64 (2H, AB system, ∆ 8.8 Hz, JAB 11.7, DMTOCH2),
2.91 (2H, AB of ABX system, ∆ 61.2 Hz, JAB 17.0, JAX 5.3,
N-4-Benzoyl-1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]cyto-
sine 9, 6,8-dioxo-3-(hydroxymethyl)-7,8,9,9a-tetrahydro-2H,6H-
pyrimido[6,1-b][1.3]oxazine 10, and N-4-benzoyl-1-(3-hydroxy-
1-methoxy-2-methylenepropyl)cytosine 7
JBX 10.3, CH C᎐O).
᎐
2
1-[3-dimethoxytrityloxy-2-(dimethoxytrityloxymethyl)prop-1-
enyl]cytosine 13
To a stirred solution of 5b (0.748 g, 1.55 mmol) in dry CH2Cl2
(30 ml) under N2 at 0 ЊC was added dropwise BCl3 (1 M in
CH2Cl2, 15 ml, 15 mmol). After stirring for 1 h at 0 ЊC, MeOH–
CH2Cl2 (1 : 1 v/v, 5 ml) was added, and the solvents removed
in vacuo. The residue was dissolved in MeOH–CH2Cl2 (1 : 1 v/v,
20 ml) and solid NaHCO3 was added to reach pH 6–7. The
solids were removed by filtration and washed with MeOH–
CH2Cl2 (1 : 1 v/v, 25 ml). The combined filtrates were concen-
trated in vacuo, and the residue partitioned between H2O (25 ml)
and CHCl3 (25 ml). Evaporation of the aqueous phase gave a
mixture of 9 and 10 as colourless crystals (0.200 g, ca. 3 : 1
estimated from 1H NMR, ca. 35% 9 and 12% 10).
To a solution of 11 (0.12 g, 0.13 mmol) in 2-PrOH (3 ml ϩ a
small amount of CH2Cl2) was added conc. aq. NH3 (3 ml),
and the solution was stirred overnight at rt, followed by con-
centration in vacuo. The residue (light yellow crystals) was used
without purification. NMR (CDCl3, selected signals): δH 7.11
(1H, d, J 7.3, H-6), 6.95 (1H, s, N–CH᎐C), 5.28 (1H, d, J 7.3,
᎐
H-5), 3.87 (2H, s, CH C᎐CH), 3.78 and 3.76 (2 × 6H, 2 × s,
᎐
2
OCH ), 3.61 (2H, s, CH C᎐CH). FABϩ MS: 802.5 (M ϩ Hϩ
᎐
3
2
calc. 802.3).
9: NMR (DMSO-d6): δH 11.28 (1H, br, NH), 8.01 (3H, d, J 7,
Ar ϩ H-6), 7.63 (1H, t, J 7, Ar), 7.52 (2H, t, J 7, Ar), 7.32 (1H,
1-[3-hydroxy-2-(hydroxymethyl)prop-1-enyl]cytosine 1b
hydrochloride
d, J 7, H-5), 6.66 (1H, br, N–CH᎐C), 5.11 (1H, t, J 4, OH), 5.04
᎐
To crude 13 (0.058 g, containing ca.0.063 mmol of 13) in
CH2Cl2 (4 ml) at rt was added dropwise HCl (4 M in dioxane)
until 13 had disappeared (TLC). Ether (1 ml) was added and
the precipitate isolated by centrifugation and washed with
CH2Cl2 and ether to give pure 1b as the hydrochloride (0.014 g,
0.06 mmol, ca. 95%) as colourless crystals. NMR (CD3OD):
(1H, br t, OH), 4.15 (2H, d, J 3.5, CH C᎐CH), 3.96 (2H, d, J 4,
᎐
2
CH C᎐CH).
᎐
2
10: An analytical sample was obtained by recrystallization
of the mixture of 9 and 10 from 2-PrOH–MeOH–ether,
mp 165–166 ЊC. NMR (DMSO-d6): δH 10.63 (1H, br, NH), 6.98
(1H, s, N–CH᎐C), 5.12 (1H, X of ABX system, JAX 5.9, JBX 9.4,
᎐
δ 7.89 (1H, d, J 7.7, H-6), 6.61 (1H, br t, N–CH᎐C), 6.10 (1H,
᎐
H
O–CH–N), 4.86 (1H, t, J 5.5, OH), 4.25 (2H, AB system, ∆ 34.3
Hz, JAB 15.8, OCH2 (ring)), 3.93 (2H, m, CH2OH), 2.83 (2H,
AB of ABX system, ∆ 20.3 Hz, JAB 16.7, JAX 5.9, JBX 9.4,
CH C᎐O). FABϩ MS: 199.0 (M ϩ Hϩ calc. 199.1) (Found:
d, J 7.7, H-5), 4.26 (2H, d, J 1.5, CH C᎐CH), 4.14 (2H, s,
᎐
2
CH C᎐CH). FABϩ MS: 198.1 (M ϩ Hϩ calc. 198.1).
᎐
2
᎐
2
6,8-Dioxo-3-(hydroxymethyl)-9-methyl-7,8,9,9a-tetrahydro-
2H,6H-pyrimido[6,1-b][1.3]oxazine 14
C, 47.8; H, 4.95; N, 13.8. Calc. for C8H10N2O4: C, 48.5; H, 5.1;
N, 14.15%).
The CHCl3 phase was dried (MgSO4), concentrated, and the
residue separated by normal gravity column chromatography,
eluted with CH2Cl2–MeOH (95 : 5 v/v), to give 9 (trace), 10
(5%), benzamide (16.5%) and 7 (20%).
7: NMR (DMSO-d6): δH 11.31 (1H, br, NH), 7.99 (2H, d, J 7,
Ar), 7.87 (1H, d, J 7, H-6), 7.62 (1H, t, J 7, Ar), 7.51 (2H, t, J 7,
Ar), 7.38 (1H, d, J 7, H-5), 6.16 (1H, s, NCH–OMe), 5.34 and
To a solution of 1a (0.054 g, 0.25 mmol) in MeOH (2 ml) was
added triethylamine (0.10 ml, 0.7 mmol). After stirring at rt
overnight the precipitate was isolated by filtration, washed with
MeOH (0.5 ml) and dried to give practically pure 14 (0.018 g,
33%) as colourless crystals. The MeOH solution contained a
ca. 55 : 45 mol% mixture of 1a and 14 (according to 1H NMR
in DMSO-d6 after removal of MeOH). An analytical sample of
14 was obtained by recrystallization from 2-PrOH, mp 197–
198 ЊC. NMR (DMSO-d6): δH 10.61 (1H, s, NH), 6.98 (1H, s,
5.16 (2 × 1H, 2 × d, J 1.2 and 1.2, C᎐CH ), 4.98 (1H, t, J 5.5,
᎐
2
OH), 3.90 (2H, m, CH2–OH), 3.30 (3H, s, O–CH3). FABϩ MS:
316.0 (M ϩ Hϩ calc. 316.1). On standing in CDCl3 solution
N-4-benzoylcytosine precipitated, and a new set of signals,
assigned the structure 8 appeared in the NMR spectrum of 7. 8:
N–CH᎐C), 4.85 (1H, t, J 5.5, OH), 4.79 (1H, d, J 9.9, O–CH–
᎐
N), 4.28 (2H, AB system, ∆ 18.4 Hz, JAB 15.7, OCH2 (ring)),
3.98–3.86 (2H, m, CH2OH), 2.81 (1H, dq, J 9.9 and 6.9,
CHCH3), 1.19 (3H, d, J 6.9, CH3). δc 169.9, 148.0, 120.0, 117.3,
83.7, 65.1, 59.8, 39.9, 10.4. FABϩ MS: 213.1 (M ϩ Hϩ calc.
NMR (CDCl3): δH 9.62 (1H, s, HC᎐O), 6.49 and 6.13 (2 × 1H,
᎐
2 × s, C᎐CH ), 4.39 (2H, s, CH –OH).
᎐
2
2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
1249

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213.1) (Found: C, 51.2; H, 5.9; N, 12.9. Calc. for C9H12N2O4: C,
50.9; H, 5.7; N, 13.2%).
176.5–178 ЊC, Rf 0.24 (EtOAc–MeOH–Et3N 90 : 8 : 2 v/v/v).
NMR (CDCl3): δH 8.32 (1H, s, H-2), 7.78 (1H, s, H-8), 7.36–
7.23 (10H, m, Ar), 6.02 (2H, br s, NH2), 4.47 (4H, s, PhCH2),
4.40 (2H, s, NCH2), 3.43 (4H, AB system, ∆ 7.8 Hz, J 9.3,
BnOCH2). δC 155.6, 152.9, 150.8, 142.4, 137.7, 128.6, 128.0,
127.9, 119.3, 74.1, 73.8, 71.8, 48.2. FABϩ MS: 420.1 (M ϩ Hϩ
calc. 420.2) (Found: C, 65.8; H, 5.9; N, 16.5. Calc. for
C23H25N5O3: C, 65.9; H, 6.0; N, 16.7%).
1-[3-Benzyloxy-2-(benzyloxymethyl)prop-1-enyl]-5-methyl-
cytosine 5c
To a stirred solution of 5a (0.988 g, 2.52 mmol) in CH3CN
(50 ml) under N2 at 0 ЊC was added Et3N (3.5 ml, 25 mmol),
1,2,4-triazole (1.76 g, 25.4 mmol), and POCl3 (0.48 ml, 5.2
mmol). Stirring was continued at 0 ЊC for 15 min and then at
rt for 4 h, the mixture was then poured into sat. aq. NaHCO3
(50 ml) and ice, and extracted with EtOAc (3 × 25 ml). The
organic phase was extracted with brine (100 ml), dried
(Na2SO4) and concentrated in vacuo. The residue was dissolved
in CH3CN (50 ml) and 25% aq. ammonia (50 ml) was added.
After stirring for 4 h at rt, brine (50 ml) was added and the
mixture was extracted with EtOAc (3 × 25 ml). The organic
phase was dried (Na2SO4), and the solvents removed in vacuo.
The solid residue was crystallized from EtOAc to give pure 5c
(0.848 g, 86%) as colourless crystals, mp 150–151 ЊC. NMR
(DMSO-d6) δH 7.42 (1H, br. s, NH), 7.38–7.25 (11H, m, Ph ϩ
N-6-(4-tert-Butylbenzoyl)-9-[3-benzyloxy-2-(benzyloxymethyl)-
2-hydroxypropyl]adenine 3d
To a stirred solution of dry 17 (13.7 g, 32.7 mmol) in dry
pyridine (200 ml) at rt was added dropwise 4-tert-butylbenzoyl
chloride (27.7 g, 141 mmol). After 2 h at rt the solution was
poured into a mixture of sat. aq. NaHCO3 (25 ml) and H2O
(225 ml), and the product extracted with EtOAc (3 × 300 ml).
To convert di- or tribenzoylated byproducts to 3d the EtOAc
solution was concentrated in vacuo and the residue dissolved in
a mixture of pyridine (325 ml), MeOH (150 ml), H2O (25 ml)
and NaOH (20 g). After 0.5 h at rt NH4Cl (32 g) was added and
solvents were removed in vacuo. The residue was treated with
CHCl3 (300 ml) and the suspension extracted with H2O (2 ×
200 ml). The organic phase was dried (Na2SO4), the solvent
removed in vacuo, and the residue purified by flash column
chromatography, eluted with CH2Cl2–MeOH 97.5 : 2.5 v/v, to
give 3d (13.9 g, 73%). A sample was crystallized from EtOAc–
heptane to give colourless crystals, mp 114.5–116 ЊC, Rf 0.57
(EtOAc–MeOH–Et3N 90 : 8 : 2 v/v/v). NMR (CDCl3): δH 9.05
(1H, br s, NH), 8.79 (1H, s, H-2), 7.97 (2H, d, J 8.5, Ar), 7.94
(1H, s, H-8), 7.53 (2H, d, J 8.5, Ar), 7.36–7.20 (10H, m, Ar),
4.49–4.40 (6H, m, PhCH2 ϩ NCH2), 3.44 (4H, s, BnOCH2),
1.36 (9H, s, tert-butyl). δC 164.5, 156.6, 152.8, 152.6, 149.6,
144.7, 137.5, 131.0, 128.6, 128.1, 127.9, 127.9, 125.9, 122.4,
73.9, 73.8, 71.7, 47.9, 35.3, 31.3. FABϩ MS: 579.9 (M ϩ Hϩ
calc. 580.3) (Found: C, 70.05; H, 6.4; N, 12.05. Calc. for
C34H37N5O4: C, 70.45; H, 6.4; N, 12.1%).
H-6), 6.92 (1H, br s, NH), 6.75 (1H, s, N–CH᎐C), 4.50 and 4.41
᎐
(2 × 2H, 2 × s, PhCH2), 4.12 and 3.97 (2 × 2H, 2 × s, BnOCH2),
1.78 (3H, s, CH3). δC 165.7, 154.6, 142.1, 138.1, 137.7, 128.6,
128.1, 128.1, 127.9, 127.6, 127.5, 127.4, 127.4, 100.9, 71.7, 71.1,
69.4, 64.0, 12.9. Fabϩ MS: 392.0 (M ϩ Hϩ calc. 392.2) (Found
C, 70.0; H, 6.5; N, 10.7. Calc. for C23H25N3O3 ϩ 1/4H2O: C,
69.8; H, 6.5; N, 10.7%).
1-[3-Hydroxy-2-(hydroxymethyl)prop-1-enyl]-5-methylcytosine
1c
To a stirred solution of 5c (0.254 g, 0.65 mmol) in dry CH2Cl2
(8 ml) at Ϫ78 ЊC under N2 was added dropwise BCl3 (1 M in
CH2Cl2, 4.0 ml, 4.0 mmol) during 15 min. Stirring was con-
tinued at Ϫ78 ЊC for 4 h, followed by dropwise addition of
MeOH–CH2Cl2 (1 : 1 v/v, 6 ml) at Ϫ78 ЊC. The cooling bath was
removed and the solvents quickly removed in vacuo to give
a residue that was immediately dissolved in MeOH–CH2Cl2
(1 : 2 v/v, 9 ml), and solid NaHCO3 was added with stirring in
small portions until the pH reached ca. 5 on wet pH paper. The
solids were removed by centrifugation and the filtrate concen-
trated in vacuo to give a brown residue that was dissolved in a
mixture of heptane (4 ml) and water (15 ml). The aqueous phase
was extracted with heptane (2 × 4 ml) followed by concen-
tration of the aq. phase in vacuo to give 1c as the hydrochloride.
This was dissolved in hot MeOH (3 ml) and a saturated solu-
tion of NaOH in MeOH was added until pH reached 9–10.
After standing overnight at Ϫ18 ЊC the crystals were isolated
by centrifugation to give pure 1c (0.096 g, 70%) as colourless
crystals, mp 229–230 ЊC (dec.). NMR (D2O) δH 7.33 (1H, s,
N-6-(4-tert-Butylbenzoyl)-9-[3-benzyloxy-2-(benzyloxymethyl)-
2-(diethoxythiophosphoryloxy)propyl]adenine 4d
To a stirred solution of dry 3d (2.89 g, 5 mmol) in dry pyridine
(20 ml) at rt under N2 was added dropwise diethyl phosphoro-
chloridite (0.94 g, 6.0 mmol). After 2 h at rt S8 (0.211 g,
6.6 mmol S) was added and the mixture stirred for another 2 h.
Pyridine was removed in vacuo and the residue in CH2Cl2
(50 ml) extracted with sat. aq. NaHCO3 (2 × 25 ml) and brine
(25 ml). The organic phase was dried (Na2SO4), the solvent
removed in vacuo, and the residue purified by flash column
chromatography, eluted with EtOAc–hexane 1 : 1 v/v, to give 4d
(3.15 g, 86%). A sample was crystallized from EtOAc–heptane
to give colourless crystals, mp 81–83 ЊC, Rf 0.59 (EtOAc–hept-
ane 8 : 2 v/v). NMR (CDCl3): δH 9.12 (1H, s, NH), 8.76 (1H, s,
H-2), 8.25 (1H, s, H-8), 7.96 (2H, d, J 8.6, Ar), 7.51 (2H, d,
J 8.6, Ar), 7.34–7.24 (10H, m, Ar), 4.80 (2H, s, NCH2), 4.46
(4H, AB system, ∆ 10.8 Hz, J 11.6, PhCH2), 4.07–3.95 (4H, m,
POCH2), 3.86 (4H, AB system, ∆ 13.2 Hz, J 10.3, BnOCH2),
1.35 (9H, s, tert-butyl), 1.18 (6H, t, J 7.0, POCCH3). δC 164.4,
156.4, 152.8, 152.6, 149.4, 144.4, 137.3, 130.9, 128.4, 127.9,
127.8, 127.7, 125.8, 122.4, 85.4 (d, JPC 9), 73.5, 69.4, 64.5 (d,
JPC 6), 45.9, 35.1, 31.1, 15.8 (d, JPC 8). δP 59.3. FABϩ MS: 732.9
(M ϩ Hϩ calc. 732.3) (Found: C, 63.2; H, 6.5; N, 8.9. Calc. for
C38H46N5O6PS: C, 62.4; H, 6.3; N, 9.6%).
H-6), 6.60 (1H, s, N–CH᎐C), 4.31 and 4.15 (2 × 2H, 2 × s, CH –
᎐
2
C᎐CH), 1.98 (3H, s, CH ). δ 166.6, 157.8, 143.6, 138.4, 125.4,
᎐
3
C
104.4, 61.0, 56.1, 12.2. Fabϩ MS: 212.1 (M ϩ Hϩ calc. 212.1)
(Found: C, 51.1; H, 6.35; N, 18.7. Calc. for C9H13N3O3 ϩ 1/
4CH3OH: C, 50.8; H, 6.4; N, 19.2%).
9-[3-Benzyloxy-2-(benzyloxymethyl)-2-hydroxypropyl]adenine
17
Dry adenine (12.5 g, 92.5 mmol) was dissolved in dry DMF
(400 ml) under N2 and NaH (60% in oil, 2.0 g, 50 mmol) added.
After stirring for 0.5 h 2 (13.9 g, 54 mmol) was added, and
the solution stirred at 110 ЊC for 3 days. The solution was
neutralized with sat. aq. NH4Cl (100 ml), diluted with H2O
(500 ml), and extracted with CHCl3 (4 × 250 ml). The combined
organic phases were extracted with brine (2 × 300 ml), dried
(Na2SO4) and concentrated in vacuo to give a yellow oil. Purifi-
cation by flash column chromatography, eluted with CH2Cl2–
MeOH 95 : 5 v/v, followed by crystallization from EtOAc–
heptane, gave pure 17 as colourless crystals (13.8 g, 60%), mp
N-6-(4-tert-Butylbenzoyl)-9-[3-benzyloxy-2-(benzyloxymethyl)-
prop-1-enyl]adenine 5d
4d (0.800 g, 1.09 mmol) Was dried by co-evaporation with dry
toluene and dissolved in dry toluene (10 ml) under N2. Dry tert-
amyl alcohol (1.2 g, 14 mmol) and NaH (60% in oil, 0.20 g,
5 mmol) were added and the mixture stirred for 3 days at rt.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
1250

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Volatiles were removed in vacuo and the residue suspended in
CH2Cl2 (50 ml) and washed with sat. aq. NaHCO3 (2 × 25 ml)
and brine (25 ml). The organic phase was dried (Na2SO4), the
solvent removed in vacuo, and the residue purified by flash
column chromatography, eluted with CH2Cl2–EtOAc 3 : 2 v/v,
to give 5d (0.740 g, 95%). A sample was crystallized from
EtOAc–heptane to give colourless crystals, mp 108.5–109.5 ЊC,
Rf 0.61 (CH2Cl2–EtOAc 3 : 2 v/v). NMR (CDCl3): δH 9.31 (1H,
s, NH), 8.76 (1H, s, H-2), 8.30 (1H, s, H-8), 7.97 (2H, d, J 8.4,
Ar), 7.50 (2H, d, J 8.4, Ar), 7.36–7.23 (10H, m, Ar), 7.21 (1H, s,
9-[3-Benzyloxy-2-(benzyloxymethyl)-2-hydroxypropyl]guanine
20
To a solution of 3-hydroxypropionitrile (8.2 ml, 120 mmol) in
dry THF (1000 ml) under N2 was added NaH (60% in mineral
oil, 5.9 g, 147 mmol). After stirring for 30 min a suspension of
19 (12.1 g, 26.7 mmol) in dry THF (100 ml) was added, and the
mixture stirred for 4 h at rt. Sat. aq. NH4Cl (250 ml) was added,
the solvents removed in vacuo, and EtOAc (200 ml) and H2O
(400 ml) added to give a precipitate that was isolated, washed
with EtOAc (100 ml) and H2O (40 ml) and dried in vacuo to give
20 (9.9 g, 85%) as a colourless solid, mp 192–194 ЊC, Rf 0.12
(EtOAc–MeOH 9 : 1 v/v). NMR (DMSO-d6, sparingly soluble):
δH 10.66 (1H, s, NH), 7.56 (1H, s, H-8), 7.34–7.23 (10H, m, Ph),
6.55 (2H, br s, NH2), 5.29 (1H, br s, OH), 4.44 (4H, s, PhCH2),
4.06 (2H, s, CH2N), 3.30 (4H, s, BnOCH2). FABϩMS: 436.2
(M ϩ Hϩ calc. 436.2) (Found: C, 59.9; H, 5.9; N, 15.6. Calc. for
C23H24ClN5O3ؒ1H2O: C, 59.7; H, 6.1; N, 15.1%).
N–CH᎐C), 4.60 (2H, s, PhCH ), 4.50 (2H, s, PhCH ), 4.27 (2H,
᎐
2
2
d, J 1.2, BnOCH2), 4.11 (2H, s, BnOCH2), 1.34 (9H, s, tert-
butyl). δC 164.6, 156.5, 153.0, 152.0, 149.8, 142.8, 137.6, 137.1,
132.2, 130.7, 128.5, 128.0, 127.9, 125.8, 122.7, 120.2, 73.3, 72.8,
70.3, 64.9, 35.1, 31.1. FABϩ MS: 562.5 (M ϩ Hϩ calc. 562.3)
(Found: C, 72.0; H, 6.2; N, 12.4. Calc. for C34H35N5O3: C, 72.7;
H, 6.3; N, 12.5%).
N-6-(4-tert-Butylbenzoyl)-9-[3-hydroxy-2-(hydroxymethyl)prop-
1-enyl]adenine 18 and 9-[3-hydroxy-2-(hydroxymethyl)prop-1-
enyl]adenine 1d
9-[3-Benzyloxy-2-(benzyloxymethyl)-2-hydroxypropyl]-N-2-
isobutyrylguanine 3e
To a solution of 20 (4.61 g, 10.6 mmol) in dry pyridine (500 ml)
was added isobutyric anhydride (5.0 ml, 30 mmol). The solution
was heated under N2 to 50 ЊC for 28 days when according to
TLC 20 was consumed. The reaction was quenched with H2O
(50 ml) and the solvents removed in vacuo. To the residue was
added EtOAc (200 ml), H2O (100ml) and sat. aq. NaHCO3
(100 ml), and the resulting precipitate was washed with H2O
(30 ml) and dried in vacuo to give 3e (3.64 g, 68%) as a colour-
less solid. A sample was recrystallized from CHCl3–heptane,
mp 204–206 ЊC, TLC Rf 0.38 (EtOAc–MeOH 9 : 1 v/v). NMR
(DMSO-d6): δH 12.04 (1H, s, NH), 11.60 (1H, s, NH); 7.85 (1H,
s, H-8), 7.32–7.26 (10H, m, Ph), 5.32 (1H, br s, OH), 4.44 (4H, s,
PhCH2), 4.18 (2H, s, CH2N), 3.35 (4H, m, BnOCH2); 2.79 (1H,
septet, J 6.5, CH3CH ), 1.11 (6H, d, J 6.5, CH3CH). δC 179.9,
154.7, 149.1, 147.4, 140.3, 137.9, 128.0, 127.4, 127.3, 119.2,
72.8, 72.6, 71.6, 47.1, 34.6, 18.9. FABϩMS: 506.1 (M ϩ Hϩ calc.
506.2).
To a stirred solution of 5d (2.70 g, 4.81 mmol) in dry CH2Cl2
(100 ml) at Ϫ78 ЊC under N2 was added dropwise BCl3 (1 M in
CH2Cl2, 28 ml, 28 mmol) during 10 min. Stirring was continued
at Ϫ78 ЊC for 4 h, followed by dropwise addition of MeOH–
CH2Cl2 (1 : 1 v/v, 50 ml) at Ϫ78 ЊC. The cooling bath was
removed and solid NaHCO3 (4.5 g) was added with stirring.
Volatiles were removed in vacuo and the solid extracted with
hexane (2 × 50 ml), followed by dry pyridine (2 × 100 ml). The
pyridine solution was evaporated, and the residue purified by
normal gravity column chromatography, eluted with toluene–
MeOH–Et3N 79 : 20 : 1 v/v/v, to give 18 (0.37 g, 20%) and 1d
(0.27 g, 25%). Both compounds contained some salts and were
further purified by recrystallization. 18, mp 235–236 ЊC (from
MeOH), Rf 0.33 (toluene–MeOH–Et3N 79 : 20 : 1 v/v/v). NMR
(DMSO-d6): δH 11.12 (1H, s, NH), 8.75 (1H, s, H-2), 8.54 (1H, s,
H-8), 8.00 (2H, d, J 8.5, Ar), 7.57 (2H, d, J 8.5, Ar), 7.02 (1H, s,
N–CH᎐C), 5.25 (1H, t, J 5.4, OH), 5.13 (1H, t, J 5.4, OH), 4.27
᎐
(2H, d, J 5.4, CH2), 4.05 (2H, d, J 5.4, CH2), 1.34 (9H, s, tert-
butyl). δC 165.34, 155.37, 152.10, 151.82, 150.36, 143.85, 139.27,
130.61, 128.35, 125.22, 124.70, 115.51, 60.87, 56.32, 34.77,
30.88. FABϩ MS: 382.3 (M ϩ Hϩ calc. 382.2) (Found: C, 62.6;
H, 6.0; N, 18.2. Calc. for C20H23N5O3: C, 63.0; H, 6.1; N, 18.4%).
1d, mp 222–223 ЊC (from H2O), Rf 0.13 (toluene–MeOH–Et3N
79 : 20 : 1 v/v/v). NMR (DMSO-d6): δH 8.23 (1H, s, H-8), 8.16
9-[3-Benzyloxy-2-(benzyloxymethyl)-2-(diethoxythiophosphoryl-
oxy)propyl]-N-2-isobutyrylguanine 4e
To a stirred solution of 3e (1.83 g, 3.61 mmol) in dry pyridine
(100 ml) under N2 was added dropwise diethyl phosphoro-
chloridite (0.57 ml, 4.0 mmol). After 30 min S8 (0.164 g,
4.0 mmol S) was added and the mixture stirred for 1 h. The
solvent was removed in vacuo, the residue dissolved in CHCl3
(80 ml), the organic phase washed with sat. aq. NaHCO3
(40 ml), dried (Na2SO4) and the solvent removed in vacuo. The
residue was crystallized from THF–heptane to give 4e (1.90 g,
80%) as colourless crystals, mp 181–183 ЊC. NMR (CDCl3):
δH 11.97 (1H, s, NH), 8.97 (1H, s, NH), 7.86 (1H, s, H-8), 7.33–
7.23 (10H, m, Ph), 4.53 (2H, s, CH2N), 4.46 (4H, s, PhCH2),
4.05–3.93 (4H, m, CH3CH2), 3.79 (4H, AB system, ∆ 4.0 Hz,
J 10.5, BnOCH2), 2.49 (1H, septet, J 7.0, CH3CH ), 1.19–1.13
(12H, m, CH3CH2 ϩ CH3CH). δC 178.6, 155.5, 149.1, 147.2,
140.4, 137.5, 128.3, 127.7, 127.5, 127.3, 120.3, 85.4 (d, JPC 9),
73.3, 69.2 (d, JPC 3), 64.6 (d, JPC 6), 45.8 (d, JPC 5), 36.3, 19.1,
15.9 (d, JPC 8). δP 59.4. FABϩMS: 658.2 (M ϩ Hϩ calc. 658.2)
(Found: C, 56.2; H, 6.1; N, 10.6. Calc. for C31H40N5O7PS:
C, 56.6; H, 6.1; N, 10.7%).
(1H, s, H-2), 7.32 (2H, br s, NH ), 6.91 (1H, br s, N–CH᎐C),
᎐
2
5.17 and 5.15 (2 × 1H, two overlapping t, J 5.5, 2 × OH), 4.22
(2H, dd, J 5.5 and 1.6, CH2), 4.01 (2H, d, J 5.5, CH2). δC 156.1,
152.9, 149.4, 140.2, 137.6, 118.1, 116.2, 61.0, 56.2. FABϩ MS:
222.1 (M ϩ Hϩ calc. 222.2) (Found: C, 48.4; H, 5.1; N, 31.2.
Calc. for C9H11N5O2 ϩ 0.1 H2O: C, 48.5; H, 5.1; N, 31.4%).
2-Amino-9-[3-benzyloxy-2-(benzyloxymethyl)-2-hydroxypropyl]-
6-chloropurine 19
To a solution of 2-amino-6-chloropurine (6.78 g, 40.0 mmol) in
dry DMF (400 ml) was added 2 (13.6 g, 48 mmol) and K2CO3
(0.60 g, 4.0 mmol), and the mixture stirred with heating to
110 ЊC under N2 for 8 h. The solvent was removed in vacuo, and
the residue purified by dry column vacuum chromatography
(0–100% EtOAc in heptane, 10% increments) to give 19 (12.1 g,
67%) as colourless crystals, Rf 0.35 (EtOAc–heptane 9 : 1 v/v),
9-[3-Benzyloxy-2-(benzyloxymethyl)prop-1-enyl]-N-2-iso-
butyrylguanine 5e
1
pure according to H NMR. A sample was recrystallized from
heptane–toluene, mp 101.5–103 ЊC. NMR (CDCl3): δH 7.74
(1H, s, H-8), 7.30–7.15 (10H, m, Ph), 4.98 (2H, s, NH2), 4.41
(4H, s, CH2Ph), 4.22 (2H, s, CH2N), 3.53 (1H, s, OH), 3.34
(4H, AB system, ∆ 12.5 Hz, JAB 9.5, BnOCH2). δC 158.8, 154.2,
151.0, 143.8, 137.2, 128.2, 127.7, 127.5, 124.5, 73.6, 73.4, 71.2,
47.2. FABϩMS: 454.3 (M ϩ Hϩ calc. 454.2) (Found: C, 60.8; H,
5.2; N, 15.3. Calc. for C23H24ClN5O3: C, 60.9; H, 5.3; N, 15.4%).
To dry tert-amyl alcohol (0.54 ml, 5.0 mmol) in dry THF
(200 ml) under N2 was added NaH (60% in oil, 0.20 g,
5.0 mmol). After 1 h was added 4e (0.63 g, 1.00 mmol, dried by
co-evaporation with dry pyridine, followed by co-evaporation
with dry toluene to remove traces of pyridine) dissolved in dry
THF (80 ml). After stirring for 27 h at rt sat. aq. NH4Cl (10 ml)
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
1251

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and H2O (25 ml) were added, and the mixture concentrated in
vacuo. The residue was dissolved in CHCl3 (100 ml) and washed
with sat. aq. NaHCO3 (2 × 30 ml), and the organic phase dried
(Na2SO4) and concentrated in vacuo. The residue was purified
by dry column vacuum chromatography (0–100% CH3CN in
toluene, 10% increments, then 0–10% MeOH in CH3CN, 1%
increments) to give 5e (0.48 g, 58%) as a colourless solid. An
analytical sample was obtained by recrystallization from THF–
heptane, mp 128–130 ЊC, Rf 0.52 (CH3CN–toluene 9 : 1 v/v).
NMR (CDCl3): δH 12.25 (1H, s, NH), 9.95 (1H, s, NH), 7.89
2,2-Bis(benzoyloxymethyl)oxirane 24
A) 3-Benzoyloxy-2-(benzoyloxymethyl)propene. 3-Chloro-
2-(chloromethyl)propene (10.04 g, 0.080 mol) was added to a
suspension of NaOBz (46.3 g, 0.320 mol) in anhydrous DMF
(350 ml). The suspension was refluxed for 4 h under N2 and
then cooled to rt. The brown suspension was filtered and the
solid washed with DMF (2 × 50 ml). The solvent was removed
in vacuo to give a brown oily residue that was dissolved in
CH2Cl2 (150 ml) and washed with brine (3 × 100 ml). The
organic phase was dried (Na2SO4), and the solvent removed
in vacuo to give the crude alkene (25 g). Purification by dry
column vacuum chromatography (0–60% EtOAc in heptane,
2% increments) gave pure 3-Benzoyloxy-2-(benzoyloxymethyl)-
propene as a colourless solid (17.48 g, 73%), mp 38–39.5 ЊC,
Rf 0.51 (hexane–CH2Cl2–EtOAc 50 : 45 : 5 v/v/v). NMR
(DMSO-d6): δH 7.97 (4H, d, J 7.4, Ar), 7.64 (2H, t, J 7.4, Ar),
(1H, s, H-8), 7.34–7.18 (10H, m, Ph), 6.83 (1H, s, N–CH᎐C),
᎐
4.48 and 4.42 (2 × 2H, 2 × s, PhCH2), 4.15 (2H, d, J 1,
BnOCH2), 3.98 (2H, s, BnOCH2), 2.76 (1H, septet, J 7,
CH3CH ), 1.17 (6H, d, J 7, CH3CH). δC 179.2, 155.4, 148.3,
147.8, 138.6, 137.3, 136.8, 131.5, 128.2, 128.1, 127.7, 127.6,
127.4, 120.2, 119.9, 76.9, 76.5, 73.1, 72.6, 70.0, 64.5, 36.0, 18.9.
FABϩMS: 488.2 (M ϩ Hϩ calc. 488.2) (Found: C, 65.0; H,
6.1; N, 14.0. Calc. for C27H29N5O4 ϩ 1H2O: C, 65.3; H, 6.1;
N, 14.1%).
7.49 (4H, t, J 7.4, Ar), 5.44 (2H, s, C᎐CH ), 4.94 (4H, s, CH ).
᎐
2
2
δC 165.3, 139.0, 133.4, 129.5, 129.2, 128.8, 116.6, 64.8. GC-MS:
296.0 (M calc. 296.1) (Found: C, 72.8; H, 5.4. Calc. for
C18H16O4: C, 73.0; H, 5.4%).
9-[3-Hydroxy-2-(hydroxymethyl)prop-1-enyl]-N-2-isobutyryl-
guanine 21
B) 2,2-Bis(benzoyloxymethyl)oxirane 24. 3-Benzoyloxy-2-
(benzoyloxymethyl)propene (5.0 g, 0.017 mol) was dissolved in
anhydrous CH2Cl2 (50 ml) and m-CPBA (70%, 6.25 g, 0.036
mol) was added. The mixture was refluxed overnight, cooled,
diluted with CH2Cl2 (50 ml) and washed with sat. aq. NaHCO3
(4 × 30 ml), 20% aq. NaHSO3 (2 × 30 ml), sat. aq. NaHCO3
(3 × 30 ml), and brine (30 ml). The organic phase was dried
(Na2SO4) and the solvent removed in vacuo to afford 24 (5.1 g,
97%) as a colourless solid, mp 60.5–61.5 ЊC, Rf 0.50 (hexane–
CH2Cl2–EtOAc 45 : 45 : 10 v/v/v). An analytical sample, crystal-
lized from EtOAc–hexane–ether, had mp 62–63 ЊC. NMR
(CDCl3): δH 8.04 (4H, d J 8.3, Ar), 7.57 (2H, t, J 7.4, Ar), 7.43
(4H, t, J 7.4, Ar), 4.51 (4H, AB system, ∆ 36 Hz, J 12.2,
BzOCH2), 2.94 (2H, s, CH2(ring)). δC 165.9, 133.2, 129.6, 129.3,
128.4, 64.4, 55.5, 49.9. FABϩMS: 313.0 (M ϩ Hϩ calc. 313.1)
(Found; C, 69.1; H, 5.15. Calc. for C18H16O5: C, 69.2; H, 5.2%).
To a stirred solution of 5e (0.460 g, 0.94 mmol) in dry CH2Cl2
(50 ml) under N2 at Ϫ78 ЊC was added BCl3 (1 M in CH2Cl2, 5.6
ml, 5.6 mmol). After 3 h at Ϫ78 ЊC a mixture of MeOH and
CH2Cl2 (1 : 1 v/v, 10 ml) was added, followed by solid NaHCO3
at Ϫ78 ЊC until neutral pH. The mixture was concentrated
in vacuo and the residue extracted first with heptane (2 × 10 ml),
then with 2-PrOH (3 × 5 ml) to give, after evaporation of
2-PrOH, crude 21 (0.29 g, 90%) as a nearly colourless solid.
NMR (CD3OD): δH 8.23 (1H, s, H-8), 6.89 (1H, s, N–CH᎐C),
4.37 (2H, d, J 1, HOCH2), 4.18 (2H, s, HOCH2), 2.72 (1H,
septet, J 6.7, CH3CH ), 1.20 (6H, d, J 6.7, CH3CH).
᎐
9-[3-Dimethoxytrityloxy-2-(dimethoxytrityloxymethyl)prop-1-
enyl]-N-2-isobutyrylguanine 22
To a solution of crude 21 (0.28 g, 0.91 mmol) in dry pyridine
(50 ml) was added 4,4Ј-dimethoxytrityl chloride (0.81 g,
2.4 mmol). After 72 h at rt the solvent was removed in vacuo
and the residue dissolved in CH2Cl2 (20 ml), washed with
sat. aq. NaHCO3 (2 × 10 ml), dried (Na2SO4) and concentrated
in vacuo. The residue was purified by dry column vacuum
chromatography (0–100% CH3CN in toluene, 10% increments)
to give 22 (0.46 g, 55%) as a colourless foam, TLC Rf 0.64
(EtOAc–MeOH 9 : 1 v/v). NMR (CDCl3): δH 7.64–7.16 (19H,
1-[3-Benzoyloxy-2-(benzoyloxymethyl)2-hydroxypropyl]thymine
25a
A mixture of thymine (1.26 g, 10 mmol) and NaH (60% in oil,
0.08 g, 2 mmol) in dry DMF (40 ml) was stirred under N2 at rt.
After 1.5 h 24 (0.625 g, 2.00 mmol) was added, and the mixture
was stirred at 110 ЊC for 24 h. After cooling to 0 ЊC sat. aq.
NH4Cl (10 ml) was added, followed by CHCl3 (50 ml) and water
(60 ml). The aqueous phase was extracted with CHCl3 (2 ×
50 ml), and the combined organic phases washed with brine,
dried (MgSO4) and evaporated in vacuo to give a light brown
oil. Purification by normal gravity column chromatography,
eluted with CH2Cl2–MeOH 97 : 3 v/v, gave the 1,3-disubstituted
thymine derivative (0.090 g, 6%) and 25a (0.640 g, 73%) as
colourless crystals. An analytical sample was obtained by re-
crystallisation from EtOAc–hexane, mp 177–178 ЊC. NMR
(CDCl3 ϩ DMSO-d6): δH 10.10 (1H, s, NH), 7.93 (4H, d, J 8,
Ar), 7.47 (2H, t, J 8, Ar), 7.33 (4H, t, J 8, Ar), 7.18 (1H, q, J 1.2,
H-6), 5.13 (1H, s, OH), 4.35 (4H, AB system, ∆ 13.5 Hz,
JAB 11.7, OCH2), 4.01 (2H, s, NCH2), 1.73 (3H, d, J 1.2, CH3).
FABϩ MS: 439.1 (M ϩ Hϩ calc. 439.1) (Found: C, 61.85; H,
5.0; N, 6.2. Calc. for C23H22N2O7 ϩ 0.5 H2O: C, 61.75; H, 5.2;
N, 6.25%).
m, H-8 ϩ Ar), 6.91–6.71 (9H, m, N–CH᎐C ϩ Ar), 4.03 (2H, s,
᎐
DMTOCH2), 3.79 and 3.76 (2 × 6H, s, 2 × OCH3), 3.68 (2H, s,
DMTOCH2), 2.60 (1H, septet, J 7.0, CH3CH ); 1.23 (6H, d,
J 7.0, CH3CH). FABϩMS: 912.3 (M ϩ Hϩ calc. 912.4).
9-[3-Hydroxy-2-(hydroxymethyl)prop-1-enyl]guanine 1e
To a solution of 22 (0.050 g, 0.055 mmol) in CH2Cl2 (5 ml) was
added sat. NH3 in MeOH (5 ml). After stirring for 48 h the
solution was concentrated in vacuo, and the residue, 23, dis-
solved in CH2Cl2 (2 ml). Addition of Cl3CCOOH in CH2Cl2
(0.20 M, 5 ml) and Et3SiH (0.044 ml, 0.28 mmol) gave a precipi-
tate that was isolated after 2 h and washed with CH2Cl2 (4 ×
1 ml). The precipitate was dissolved in H2O (2 ml) and 1e was
precipitated from the aqueous solution by neutralization with
1 M aq. NaOH. The precipitate was washed with H2O followed
by ether to give 1e (0.009 g, 70%) as colourless crystals, mp.
ca. 265 ЊC (dec.). NMR (DMSO-d6): δH 10.63 (1H, s, NH);
1-[2,3-Dihydroxy-2-(hydroxymethyl)propyl]thymine 26a
7.79 (1H, s, H-8), 6.72 (1H, s, N–CH᎐C), 6.52 (2H, br s, NH ),
25a (0.140 g, 0.32 mmol) Was dissolved in a mixture of MeOH
(2 ml) and aq. NH3 (32%, 2 ml). After 24 h at rt the solution was
concentrated in vacuo, H2O (15 ml) was added, the mixture
extracted with CHCl3 (3 × 10 ml), and the aqueous phase
was concentrated in vacuo. Recrystallization from 2-PrOH gave
26a (0.053 g, 72%) as colourless crystals, mp 189–192 ЊC. NMR
᎐
2
5.11 and 5.03 (2 × 1H, 2 × t, J 5.3 and 5.3, 2 × OH), 4.17 (2H, d,
J 5.3, HOCH2), 3.98 (2H, d, J 5.3, HOCH2). δC 156.7, 153.8,
151.1, 137.0, 136.7, 116.3, 116.0, 60.9, 56.1. FABϪ MS: 236.1
(M Ϫ Hϩ calc. 236.1) (Found: C, 45.4; H, 4.5; N, 28.8. Calc. for
C9H11N5O3 ϩ 1H2O: C, 45.6; H, 4.7; N, 29.5%).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 2 4 5 – 1 2 5 4
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(DMSO-d6): δH 11.26 (1H, s, NH), 7.40 (1H, s, H-6), 4.56 (2H, t,
1/2(JAXϩJBX) 5.7, OH), 4.53 (1H, s, OH), 3.71 (2H, s, NCH2),
3.26 (4H, AB of an ABX system appearing as a doublet,
OCH2), 1.74 (3H, s, CH3). δC 164.2, 152.1, 143.0, 107.7, 74.7,
62.9, 49.7, 12.1. FABϩ MS: 231.0 (M ϩ Hϩ calc. 231.1) (Found:
C, 46.7; H, 5.95; N, 12.2. Calc. for C9H14N2O5: C, 46.95; H, 6.1;
N, 12.15%).
concentrated in vacuo. Recrystallization from 2-PrOH–H2O
gave 26c (0.058 g, 84%) as colourless crystals, mp 220–221 ЊC
(lit.20 mp 218–220 ЊC). NMR (DMSO-d6): δH 8.13 (1H, s, H-2),
7.99 (1H, s, H-8), 7.27 (2H, s, NH2), 4.87 (2H, t, 1/2(JAXϩJBX
)
6.0, OH), 4.75 (1H, s, OH), 4.18 (2H, s, NCH2), 3.24 (4H, AB of
an ABX system, ∆ 14.7 Hz, JAB 11.2, JAX 6.1, JBX 5.9, OCH2).
δC 156.0, 152.2, 150.0, 142.2, 118.1, 74.3, 62.9, 46.1. FABϩ MS:
240.1 (M ϩ Hϩ calc. 240.1) (Found: C, 45.3; H, 5.45; N, 29.6.
Calc. for C9H13N5O3: C, 45.2; H, 5.5; N, 29.3%).
N-4-Benzoyl-1-[3-benzoyloxy-2-(benzoyloxymethyl)2-hydroxy-
propyl]cytosine 25b
2-Amino-9-[3-benzoyloxy-2-(benzoyloxymethyl)-2-hydroxy-
propyl]-6-chloropurine 25d
A mixture of N-4-benzoylcytosine (0.86 g, 4.0 mmol) and NaH
(60% in oil, 0.08 g, 2 mmol) in dry DMF (20 ml) was stirred
under N2 at rt. After 1.5 h 24 (0.600 g, 1.90 mmol) was added,
and the mixture was stirred at 110 ЊC for 24 h. After cooling
to 0 ЊC sat. aq. NH4Cl (10 ml) was added, followed by CHCl3
(50 ml) and H2O (60 ml). The aqueous phase was extracted with
CHCl3 (2 × 50 ml), and the combined organic phases were
washed with brine, dried (MgSO4) and evaporated in vacuo to
give almost pure 25b (0.740 g, 74%) as colourless crystals.
Recrystallization from EtOAc–MeOH gave pure 25b (0.65 g,
65%), mp 200–201 ЊC. NMR (CDCl3 ϩ DMSO-d6): δH 9.58
(1H, s, NH), 7.93 (4H, d, J 8, Ar), 7.86 (2H, d, J 8, Ar), 7.83
(1H, d, J 7.3, H-6), 7.52–7.28 (10H, m, Ar ϩ H-5), 5.59 (1H, s,
OH), 4.43 (4H, AB system, ∆ 18.2 Hz, JAB 11.8, OCH2), 4.30
(2H, s, NCH2). FABϩ MS: 528.3 (M ϩ Hϩ calc. 528.2) (Found:
C, 65.9; H, 4.65; N, 7.9. Calc. for C29H25N3O7: C, 66.0; H, 4.8;
N, 7.95%).
A mixture of 2-amino-6-chloropurine (0.51 g, 3 mmol) and
K2CO3 (0.04 g, 0.3 mmol) in dry DMF (15 ml) was stirred
under N2 at rt. After 1.5 h 24 (0.600 g, 1.90 mmol) was added,
and the mixture was stirred at 105 ЊC for 6 h. After cooling, the
mixture was filtered and evaporated in vacuo to give light brown
crystals. Purification by normal gravity column chromato-
graphy, eluted with CH2Cl2–MeOH 95 : 5 v/v, gave first the N-7-
isomer (0.14 g, 9.7%) and then 25d (0.93 g, 64%) as colourless
crystals, mp 146–148 ЊC. NMR (CDCl3): δH 7.99 (4H, d, J 8,
Ar), 7.89 (1H, s, H-8) 7.59 (2H, t, J 8, Ar), 7.44 (4H, t, J 8, Ar),
4.99 (2H, s, NH), 4.76 (1H, br s, OH), 4.46 (2H, s, NCH2), 4.44
(4H, AB system, ∆ 14.7 Hz, JAB 11.8, OCH2). FABϩ MS: 482.2
(M ϩ Hϩ calc. 482.1) (Found: C, 57.05; H, 4.05; N, 14.2. Calc.
for C23H20ClN5O5: C, 57.35; H, 4.2; N, 14.55%).
9-[2,3-Dihydroxy-2-(hydroxymethyl)propyl]guanine 26d
1-[2,3-Dihydroxy-2-(hydroxymethyl)propyl]cytosine 26b
25d (0.400 g, 0.83 mmol) Was refluxed with aq. HCl (2 M,
15 ml). After 4 h the solution was cooled and extracted with
CHCl3 (3 × 10 ml), and the aqueous phase was evaporated
in vacuo to give the hydrochloride of 26d as colourless crystals
(0.243 g, 100%). The hydrochloride was dissolved in water
(10 ml), aq. NaOH (2 M) was added to pH ca. 8, where upon
26d (0.125 g, 59%) precipitated as colourless crystals. An ana-
lytical sample was obtained by recrystallization from 2-PrOH–
H2O, mp > 300 ЊC. NMR (DMSO-d6): δH 10.59 (1H, s, NH),
25b (0.450 g, 0.85 mmol) Was dissolved in a mixture of MeOH
(5 ml) and aq. NH3 (32%, 5 ml). After 24 h at rt the solution was
concentrated in vacuo, water (15 ml) was added, the mixture
extracted with CHCl3 (3 × 10 ml), and the aqueous phase was
concentrated in vacuo. Recrystallisation from 2-PrOH–H2O
gave 26b (0.144 g, 73%) as colourless crystals, mp 228–229 ЊC.
NMR (DMSO-d6): δH 7.48 (1H, d, J 7.0, H-6), 7.18 and 7.13
(2 × 1H, 2 × br s, NH2), 5.70 (1H, d, J 7.0, H-5), 4.80 (1H, s,
OH), 4.79 (2H, t, 1/2(JAXϩJBX) 6.2, OH), 3.73 (2H, s, NCH2),
3.20 (4H, AB of an ABX system, ∆ 23.5 Hz, JAB 11.4, JAX 7.0,
JBX 5.5, OCH2). δC 166.0, 158.0, 147.7, 93.4, 74.9, 63.1, 51.2.
FABϩ MS: 216.1 (M ϩ Hϩ calc. 216.1) (Found: C, 41.2; H, 6.5;
N, 17.8. Calc. for C8H13N3O4 ϩ H2O: C, 41.2; H, 6.5; N, 18.0%).
7.56 (1H, s, H-8), 6.53 (2H, s, NH), 4.75 (2H, t, 1/2(JAXϩJBX
)
6.1, OH), 4.68 (1H, s, OH), 3.97 (2H, s, NCH2), 3.22 (4H, AB
of an ABX system, ∆ 12.6 Hz, JAB 11.4, JAX 6.2, JBX 5.9, OCH2).
δC 157.0, 153.5, 151.7, 139.2, 115.9, 74.3, 62.8, 45.9. FABϩ MS:
256.0 (M ϩ Hϩ calc. 256.1) (Found: C, 38.45; H, 5.5; N, 24.7.
Calc. for C9H13N5O4ؒ1.5 H2O: C, 38.3; H, 5.7; N, 24.8%).
N-6-Benzoyl-9-[3-benzoyloxy-2-(benzoyloxymethyl)2-hydroxy-
propyl]adenine 25c
Acknowledgements
A mixture of N-6-benzoyladenine (0.96 g, 4.0 mmol) and NaH
(60% in oil, 0.08 g, 2 mmol) in dry DMF (20 ml) was stirred
under N2 at rt. After 1.5 h 24 (0.600 g, 1.90 mmol) was added,
and the mixture stirred at 110 ЊC for 24 h. After cooling to 0 ЊC
sat. aq. NH4Cl (10 ml) was added, followed by CHCl3 (50 ml)
and H2O (60 ml). The aqueous phase was extracted with CHCl3
(2 × 50 ml), and the combined organic phases were washed with
brine, dried (MgSO4) and evaporated in vacuo to give a light
brown oil. Purification by normal gravity column chromato-
graphy, eluted with CH2Cl2–MeOH 97 : 3 v/v gave first the N-7-
isomer (ca. 10%) and then 25c (0.452 g, 43%) as colourless
crystals, mp 193–195 ЊC. NMR (CDCl3): δH 9.2 (1H, br, NH),
8.58 (1H, s, H-2), 8.29 (1H, s, H-8), 7.99–7.28 (15H, m, Ar),
5.61 (1H, s, OH), 4.63 (2H, s, NCH2), 4.37 (4H, AB system,
∆ 38.1 Hz, JAB 11.7, OCH2). FABϩ MS: 552.3 (M ϩ Hϩ calc.
552.2) (Found: C, 64.85; H, 4.45; N, 12.55. Calc. for
C30H25N5O6: C, 65.3; H, 4.55; N, 12.7%).
Lotte-Emilie Larsen is thanked for the preparation of 24. The
Danish Natural Science Research Council and The Danish
Technical Research Council are thanked for financial support.
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