Synthesis of various 5-alkoxymethyluracil analogues and structure-cytotoxic activity relationship study

Article abstract of DOI:10.1016/j.carres.2011.07.026

A number of 5-alkoxymethyluracil analogues were synthesized to evaluate their cytotoxic activity. 5-Alkoxymethyluracil derivatives 1 were prepared via known nucleophilic substitution of 5-chloromethyluracil 5 and subsequently transformed to their correspo

Full text of DOI:10.1016/j.carres.2011.07.026

Carbohydrate Research 346 (2011) 2136–2144
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of various 5-alkoxymethyluracil analogues
and structure–cytotoxic activity relationship study
a
b
b
ˇ
ˇ a,
⇑
Lucie Brulíková , Petr Dzubák , Marián Hajdúch , Jan Hlavác
a
´
Department of Organic Chemistry, Faculty of Science, Palacky University, 17 Listopadu 12, Olomouc 771 46, Czech Republic
b Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacky´ University and University Hospital in Olomouc,
Puškinova 6, Olomouc 775 20, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2011
Received in revised form 28 July 2011
Accepted 31 July 2011
Available online 5 August 2011
A number of 5-alkoxymethyluracil analogues were synthesized to evaluate their cytotoxic activity. 5-Alk-
oxymethyluracil derivatives 1 were prepared via known nucleophilic substitution of 5-chloromethylura-
cil 5 and subsequently transformed to their corresponding nucleosides 2. All prepared compounds were
submitted to cytotoxic activity testing against drug sensitive and drug resistant leukaemia cells and solid
tumour derived cell lines. In addition, the cytotoxic activity of 5-alkoxymethyluracil analogues 1 and 2
was compared with the previously published 5-[alkoxy(4-nitrophenyl)methyl]uracil analogues 3 and
4. Extensive structure–cytotoxic activity relationship studies are reported.
Keywords:
Nucleosides
Alkoxymethyluracils
Alkoxymethyluridines
Cytotoxic activity
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Despite the fact that many published studies describe synthesis
and biological activity of various 5-alkoxymethyluracils (1a–o,
Various 5-alkoxymethyluracil analogues have been studied over
the last 60 years in connection with either their synthesis or nu-
cleic acid metabolism. The oldest work describes the synthesis of
5-alkoxymethyluracil analogues via formaldehyde addition,1 from
5-bromomethyl and 5-chloromethyluracil or 5-hydroxymethyl-
uracil.2–4 However, intensive efforts to find new successful drugs
of antimetabolite type have led, among others, to studies focused
on the potential antiviral and cytotoxic activity of 5-alkoxymethyl-
uracil analogues.
Fig. 1) with alkyl chain length C1–C12 (except C9 and C11),1–3,5,10,11
5-alkoxymethyluridines with alkyl chains longer than C2 were not
studied to date.1,12,13 Herein, we report a relatively facile and effi-
cient method for the synthesis of various 5-alkoxymethyluridines
with a C3–C12 chain length (2c–2o, Fig. 1). Because little is known
about anticancer studies of the 5-alkoxymethyluracil analogues 1
and 2, this work also demonstrates the cytotoxic potential of 5-alk-
oxymethyluracil analogues 1 and 2 in vitro against cancer cells of
different histogenetic origin, genetic background and drug resis-
tance profile.
In earlier studies we have also reported on the cytotoxic activity
and structure–activity relationship of 5-[alkoxy(4-nitrophenyl)
methyl]uracils 314 and 5-[alkoxy(4-nitrophenyl)methyl] uridines
4f–i15 (Fig. 2). In the context of the current paper, the relationship
between structure and cytotoxic activity is derived from 5-alk-
oxymethyluracils 1, 5-alkoxymethyluridines 2, 5-[alkoxy(4-nitro-
To illustrate the antiviral activity of 5-alkoxymethyl derivatives
derived from 20,30-dideoxyuridine and 20,30-dideoxycytidine ana-
logues,5 5-alkoxymethyl-30-azido-20,30-dideoxyuridines,6 30-ami-
no-20,30-dideoxyuridine7 or agents with an ethoxymethyl side
chain at C-5 and an ethyl group at the N-1 position of the uracil
ring8 or 5-alkoxymethylazidothymidine analogues and their corre-
sponding a
-anomers9 were reported. On the other hand, research
on new anticancer agents in the field of 5-alkoxymethyluracil ana-
logues has attracted much less attention. One of the few examples
is the 5-alkoxymethyl-30-amino-20,30-dideoxynucleosides tested
against human epidermoid cervical cancer cells.7
phenyl)methyl]uracils 3 and 5-[alkoxy(4-nitrophenyl)methyl]
uridines 4 to evaluate the effect of the 4-nitrophenylmethyl group
in position 5 and the ribose moiety in position 1.
A considerable part of this work is based on study of the chem-
ical reactivity of the hydroxymethyl group of 5-hydroxymethylura-
cil and its conversion to the corresponding 5-alkoxymethyluracil.
2. Results and discussion
2.1. Synthesis
In the work reported here, a series of 5-alkoxymethyluracil ana-
logues 1 and 2 were prepared via nucleophilic substitution of 5-
⇑
Corresponding author. Tel.: +420 585 634 405; fax: +420 585 634 465.
ˇ
E-mail address: hlavac@orgchem.upol.cz (J. Hlavác).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.07.026

L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
2137
O
R
O
h) R = -(CH2)7CH3
i) R = -(CH2)8CH3
j) R = -(CH2)9CH3
k) R = -(CH2)10CH3
l) R = -(CH2)11CH3
m) R = -i-Pr
HN
O
a) R = -CH3
R
b) R = -CH2CH3
c) R = -(CH2)2CH3
d) R = -(CH2)3CH3
e) R = -(CH2)4CH3
f) R = -(CH2)5CH3
g) R = -(CH2)6CH3
HN
O
O
N
HO
O
N
H
O
OH OH
1
n) R = -s-But
2
o) R = -t-But
Figure 1. 5-Alkoxymethyluracils 1 and 5-alkoxymethyluridines 2.
NO2
NO2
h) R = -(CH2)7CH3
a) R = -CH3
i) R = -(CH2)8CH3
j) R = -(CH2)9CH3
k) R = -(CH2)10CH3
l) R = -(CH2)11CH3
m) R = -i-Pr
b) R = -CH2CH3
c) R = -(CH2)2CH3
d) R = -(CH2)3CH3
e) R = -(CH2)4CH3
f) R = -(CH2)5CH3
g) R = -(CH2)6CH3
O
R
O
HN
O
R
HN
O
O
N
HO
O
N
H
n) R = -s-But
o) R = -t-But
O
3
OH OH
4
Figure 2. 5-[Alkoxy(4-nitrophenyl)methyl]uracils 3 and 5-[alkoxy(4-nitrophenyl)methyl]-uridines 4.
chloromethyluracil 5. This starting material was synthesized by a
well-known and described reaction2 from 5-hydroxymethyluracil.
Subsequently, this reactive intermediate was heated at 100 °C in
various aliphatic linear alcohols C1–C12 as well as in selected
branched alcohols such as isopropanol, sec-butanol and tert-buta-
nol. However, only two of this series, 5-nonyloxymethyluracil
(1i) and 5-undecyloxymethyluracil (1k) have not yet been de-
scribed and their preparation is included in Section 4.
First, the cytotoxic activity of modified nucleobases 1 was
investigated against a diverse range of cancer cells. All results are
reported in Table 1. While derivatives with a short linear as well
as branched side chain exhibit no significant activity against any
of the tested cell lines, interesting dependences on the length of
the alkyl chain is observed in comparison of derivatives 1a–l. All
those activities were affected by varying the alkyl chain and they
are illustrated in Figure 3.
All prepared alkoxymethyluracils 1 were silylated in 1,1,1,3,3,3-
hexamethyldisilazane (HMDS) at reflux with addition of (NH4)2SO4
as the catalyst (Scheme 1). Silylated derivatives 6 were coupled
It is evident that the maximum cytotoxicity is observed for
derivatives with an alkyl chain length of between 6 and 9 methy-
lene groups excepting drug resistant lines K562-tax and CEM-DNR-
bulk. However, another carbon addition leads to marked decrease
in cytotoxic activity. Although none of derivatives 1a–l exhibit
interesting activity against CEM, K562, A562, HCT116p53 or
HCT116p53ꢀ/ꢀ cancer cell lines, more interesting results were
obtained in case of drug resistant leukaemia cell lines (CEM-
DNR-bulk and K562-tax), where derivatives 1 exhibit activity in
low-micromolar concentrations (CEM-DNR-bulk, derivative 1k:
with 1-O-acetyl-20,30,50-tri-O-benzoyl-b-
D-ribofuranose 7 using tri-
methylsilyl trifluoromethanesulfonate as the Lewis acid. This so-
called Vorbrügen method16 led to the synthesis of protected
nucleosides 8, which were successfully purified using silica gel col-
umn chromatography to afford the parent nucleosides in 34–69%
yields. Treatment of benzoylated nucleosides 8 with an excess of
methanolic ammonia solution gave a series of fifteen alkoxynucle-
osides 2, where derivatives (2c–o) are newly synthesized com-
pounds (Scheme 1).
relative IC50 = 2.4 lM). Figure 3 shows the selectivity of tested
compounds 1 against CEM-DNR-bulk cell line, where the cytotoxic
effect is directly proportional to the increasing length of the alkyl
chain. On the other hand, the trend of increasing cytotoxicity
against the K562-tax cell line with growing number of carbons in
alkyl chain ends with 11 methylene groups. The evidence of
increasing cytotoxic activity specifically in drug resistant cell lines
but not in drug sensitive counterparts with growing length of alkyl
chain might indicate interaction of long lipophilic groups with
multidrug resistance transporters such as P-glycoprotein or the
multidrug resistance-associated protein-1 (MRP).
On the other hand, one might suppose that derivatives 1, as well
as the majority of antimetabolites, might inhibit DNA and/or RNA
synthesis, for instance, via incorporation into the nucleic acids, ar-
rest cell division and induce apoptosis. Therefore, the modified
nucleobases 1 were transformed to the corresponding nucleosides
2. Moreover, introduction of the ribose moiety positively affects
the solubility of compounds in water. The activities of nucleosides
2 are summarized in Table 2 and illustrated in Figure 4.
2.2. Cytotoxic activity and structure–activity relationship
All compounds prepared within this paper were tested under
in vitro conditions for their cytotoxic activity against cancer cell
lines CEM, K562, their drug resistant counterparts CEM-DNR-bulk
and K562-tax, and A549, HCT116p53 and HCT116p53ꢀ/ꢀ cell
lines. In previous work we have also demonstrated the cytotoxic
activity of various 5-[alkoxy(4-nitrophenyl)methyl]uracils 314
and 5-[alkoxy(4-nitrophenyl)methyl]uridines 4.15 Within the
structure–cytotoxic activity relationship studied in this paper, we
extended the cytotoxic studies of derivatives 3 and 4 and tested
their activity for two more cancer cell lines (HCT116p53 and
HCT116p53ꢀ/ꢀ). The presence of the nitrophenyl moiety offers
an interesting opportunity to study the relationships between the
structure of 5-alkoxymethyluracils 1, 5-alkoxymethyluridines 2,
5-[alkoxy(4-nitrophenyl)methyl]uracils 3 and 5-[alkoxy(4-nitro-
phenyl)methyl]uridines 4, the various lengths of the alkoxy side
chain versus cytotoxic activity.
In contrast to bases 1 it is apparent that the most active nucleo-
sides 2 are those with a longer alkyl chain containing 9–11 meth-

2138
L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
O
O
OSi(CH3)3
N O
R
R
HN
Cl
ROH
100°C
HMDS
(NH4)2SO4
HN
O
O
N
H
5
O
N
H
1
(H3C)3SiO
N
140°C
6
a) R = -CH3
b) R = -CH2CH3
c) R = -(CH2)2CH3
BzO
OAc
O
TMSOTf
RT
d) R = -(CH2)3CH3
e) R = -(CH2)4CH3
f) R = -(CH2)5CH3
g) R = -(CH2)6CH3
h) R = -(CH2)7CH3
i) R = -(CH2)8CH3
j) R = -(CH2)9CH3
k) R = -(CH2)10CH3
l) R = -(CH2)11CH3
m) R = -i-Pr
OBz OBz
7
O
N
O
N
R
R
HN
O
HN
O
O
O
MeOH/NH3
RT
HO
BzO
O
O
n) R = -s-But
o) R = -t-But
OH OH
OBz OBz
2
8
Scheme 1. General synthetic route of the lead compounds 2.
Table 1
Summary of cytotoxic activity of derivatives 1a
Compound code
R
K562
K562-tax
CEM
CEM-DNR-bulk
HCT116p53
HCT116p53ꢀ/ꢀ
A549
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
CH3
CH2CH3
192.6
188.1
189.2
174.9
199.7
149.4
74.1
134.3
133.7
115.9
74.0
148.8
129.7
50.8
46.2
22.8
9.3
6.8
85.2
75.3
77.1
77.1
104.7
74.3
51.8
53.5
55.0
77.6
73.0
92.4
85.2
85.9
102.9
53.1
60.7
58.4
28.3
65.1
39.0
26.2
18.0
6.1
4.9
2.4
2.4
55.3
42.1
132.1
200.0
200.3
196.6
214.4
223.4
168.7
83.3
136.0
211.1
227.4
226.7
225.5
190.5
191.8
185.9
191.5
193.8
187.2
207.6
198.2
148.6
91.7
107.8
148.5
173.1
176.5
185.1
192.8
175.6
215.5
244.6
241.7
228.4
231.4
248.1
217.1
167.2
151.4
245.5
249.1
248.0
243.3
228.8
243.2
160.6
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
i-Pr
48.8
109.1
151.0
163.8
169.5
176.5
188.8
162.6
30.5
131.9
131.3
163.7
s-But
t-But
a
Average values of IC50 from 3 to 4 independent experiments with SD ranging from 10% to 25% of the average values.
cates significantly decreased activity of nucleosides 2. Cytotoxic
activities of compounds 1 and 2 in drug sensitive versus MDR po-
sitive cell lines collectively suggest the importance of lipophilic
groups for interaction of the compounds with ATP-dependent
MDR transporters.
The next aim of our study was to investigate the role of substi-
tution of nucleobases 1 in position C-5 with the bulky nitrophenyl
group. This group causes a rise of chirality of the substituent in po-
sition 5. Because of the impossibility of separating the enantio-
mers, the compounds 3 were tested in their racemic mixture.14
Indeed, comparison of cytotoxic activity of bases 1 and 3 (Tables
1 and 3) indicates that substitution of the methylene group by
the nitrophenyl moiety positively influences the cytotoxic activity
against CEM, A549, K562 and both colorectal carcinoma cell lines.
On the other hand, the activity of bases 3j–3k against P-gp positive
K562-tax cell line is diminished in comparison to active analogues
1j–1k. Also the activity against the MRP-1 positive CEM-DNR-bulk
line is diminished namely in case of derivatives 1i–1l, suggesting
variable activity in those two MDR transporters.
Figure 3. Cytotoxic activity of compounds 1 as a function of chain length in
different cell lines.
ylene groups. While the activity of nucleosides 2a–h against CEM,
A549, K562 and both colorectal carcinoma cell lines is approaching
the activity of bases 1a–h, nucleosides 2i–l exhibit much better
activity than bases 1i–l. Most interestingly, a comparison of cyto-
toxic activity of nucleosides 2 and bases 1 against drug resistant
leukaemia cancer cell lines (CEM-DNR-bulk and K562-tax) indi-
As mentioned above, a detailed study of the structure–activity
relationship was complicated due to the chirality of the carbon in
position C-5 of uracil derivatives 3. Because the corresponding
enantiomers 3 are impossible to separate, they were converted to

L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
2139
Table 2
Summary of cytotoxic activity of derivatives 2a
Compound code
R
K562
K562-tax
CEM
CEM-DNR-bulk
HCT116p53
HCT116p53ꢀ/ꢀ
A549
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
CH3
CH2CH3
181.5
180.1
180.3
203.8
192.2
138.3
134.5
70.2
38.8
13.6
13.6
35.1
194.6
183.0
180.4
196.8
180.1
173.0
167.3
153.3
109.6
94.5
74.0
72.1
183.0
192.2
186.1
92.2
98.0
180.6
170.1
178.6
155.4
174.6
173.5
176.2
137.9
62.7
54.0
82.2
88.6
173.7
172.7
208.3
183.8
187.4
177.5
193.5
184.5
174.8
142.6
117.0
118.4
64.9
90.4
84.9
196.8
199.3
192.5
210.1
220.5
229.2
210.7
199.5
204.1
195.8
165.1
144.6
97.5
121.5
117.5
205.6
215.8
205.7
160.7
165.4
147.5
181.2
180.6
166.5
166.1
138.2
104.3
41.8
17.8
41.4
168.8
180.4
177.2
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
i-Pr
100.7
93.3
100.2
109.6
81.3
82.4
52.5
35.3
28.0
31.2
103.2
94.8
83.1
179.6
207.9
192.4
s-But
t-But
a
Average values of IC50 from 3 to 4 independent experiments with SD ranging from 10% to 25% of the average values.
When bases 3 and ribosides 4 are compared, a slight increase of
activities can be defined in case of some nucleosides, especially for
derivative 4i, when a micromolar value of IC50 against CEM and
HCT116p53 was reached.
3. Conclusion
In summary, 5-alkoxymethyluracil analogues were synthesized
employing Vorbrügen ribosylation. The cytotoxic activity of both
5-alkoxymethyluracils 1 and 5-alkoxymethyluridines 2 was evalu-
ated and extensive SAR studies are reported. The present SAR stud-
ies illustrate that modified nucleobases 1 are more active against
multidrug drug resistant K562-tax and CEM-DNR-bulk cell lines
in direct comparison with corresponding nucleosides 2, whereas
nucleosides 2 exhibit higher activity against drug sensitive cell
lines, especially longer alkyl chain derivatives 2i–l with cytotoxic
activity in low micromolar concentrations. Compounds 1i–l and
3j–l exhibited substantial selectivity against chemoresistant line
K562-tax (P-glycoprotein positive), while in CEM-DNR-bulk cells
(MRP-1 positive), selective activity was seen only in compounds
1i–l. This interesting phenomenon will be a subject of mechanistic
study in the future. Introduction of the nitrophenyl moiety en-
hanced cytotoxicity of the bases 3 in comparison to 1 with the
exception of the multidrug resistant CEM-DNR-bulk cell line sug-
gesting that nitrophenyl moiety is involved in MRP-1 mediated
drug efflux from the cell. While the presence of a ribose moiety
in the structure of derivatives 4 modulates in vitro anticancer po-
Figure 4. Cytotoxic activity of compounds 2 as a function of chain length in
different cell lines.
corresponding diastereoisomeric nucleosides 4(+) and 4(ꢀ). Activ-
ities of model diastereoisomers 4(+) and 4(ꢀ) (Table 4) were com-
pared in order to identify any possible effect of chirality for
compounds bearing appropriate chiral centres. However, the trend
of activity was similar for both diastereoisomers 4(+) as well as for
4(ꢀ) and the cytotoxic potencies of (+) and (ꢀ) diastereoisomers
were similar regardless of their chain length. Therefore, we con-
clude that the chirality of carbon in position C-5 of uracil does
not influence the cytotoxic activity of the tested compounds.
Table 3
Summary of cytotoxic activity of derivatives 3a
Compound code
R
K562
K562-tax
CEM
CEM-DNR-bulk
HCT116p53
HCT116p53ꢀ/ꢀ
A549
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
CH3
CH2CH3
187.4
168.0
139.0
116.5
64.9
118.5
34.4
38.7
177.3
172.9
135.8
111.6
81.7
91.3
37.7
44.2
31.3
23.5
26.2
14.1
172.7
161.1
175.5
134.1
128.6
49.9
57.3
49.1
113.7
28.8
24.3
25.2
22.3
20.5
14.2
124.7
103.0
131.1
169.9
171.8
151.8
144.5
116.7
137.7
44.1
46.9
48.9
63.9
96.0
166.1
158.2
110.2
87.4
54.5
70.4
33.0
33.7
27.7
26.7
163.3
149.8
112.7
109.3
88.0
26.3
41.0
40.5
41.3
40.3
50.4
23.5
143.4
141.5
158.5
158.2
151.7
126.0
115.2
78.8
83.0
39.7
39.3
42.2
67.7
95.8
120.1
148.5
137.8
161.5
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)10CH3
(CH2)11CH3
i-Pr
36.1
78.7
118.0
47.7
172.4
148.4
175.0
68.5
114.3
169.6
165.3
162.8
166.0
155.1
136.7
178.5
s-But
t-But
a
Average values of IC50 from 3 to 4 independent experiments with SD ranging from 10% to 25% of the average values. Compounds 3 were tested in their racemic mixture.

2140
L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
Table 4
Summary of cytotoxic activity of derivatives 4a
Compound code
R
K562
K562-tax
CEM
CEM-DNR-bulk
HCT116p53
HCT116p53ꢀ/ꢀ
A549
4f (+)
4f (ꢀ)
4g (+)
4g (ꢀ)
4h (+)
4h (ꢀ)
4i (+)
(CH2)5CH3
(CH2)5CH3
(CH2)6CH3
(CH2)6CH3
(CH2)7CH3
(CH2)7CH3
(CH2)8CH3
(CH2)8CH3
58.4
64.5
33.6
36.7
15.1
24.7
10.9
13.0
141.4
136.9
42.8
52.3
33.1
34.4
14.6
23.9
39.8
45.5
12.8
29.0
11.4
16.9
7.9
131.0
145.8
103.7
95.6
34.6
36.6
53.0
51.9
24.8
30.0
15.0
15.5
9.0
104.4
105.0
38.5
52.8
28.1
30.7
15.8
17.3
121.3
156.0
39.8
69.9
38.7
39.7
23.1
29.1
32.3
33.8
4i (ꢀ)
10.0
9.8
a
Average values of IC50 from 3 to 4 independent experiments with SD ranging from 10% to 25% of the average values.
tency of the compounds, their chirality had no impact. We expect
that our SAR studies will be extended in future. Particularly, we
would like to examine the cytotoxicity of derivatives 1 and 2 with
alkyl chains longer than 12 carbons in drug resistant cancers,
which currently represent the major problem in successful anti-
cancer therapy.
109.80, 140.85, 151.84, 164.32. MS m/z calcd for C16H28N2O3:
296.41, found 295.13 [MꢀH]ꢀ.
4.1.3. General procedure for preparation of 5-alkoxymethyl-1-
(20,30,50-tri-O-benzoyl-b-
D-ribofuranosyl)pyrimidine-2,4(1H,3H)-
diones 8
Uracils 1 were heated at reflux in hexamethyldisilazane with
(NH4)2SO4 (approximately 5 mg) for 8 h. The solution was the con-
centrated and the residue was dissolved in anhydrous 1,2-dichlo-
roethane (20 mL). To this solution, 1-O-acetyl-20,30,50-tri-O-
4. Experimental section
4.1. Chemistry
benzoyl-b-D-ribofuranose and trimethylsilyl trifluoromethanesul-
fonate were added. This mixture was stirred at room temperature
for 24 h, washed with water (20 mL) and EtOAc (3 ꢁ 60 mL), dried
over sodium sulfate, filtered and evaporated to dryness. The crude
product was purified by preparative chromatography using CHCl3–
CH3CN (5:1) to get the nucleosides 8.
Melting points were determined on a Boetius stage and are
uncorrected. NMR spectra were recorded at ambient temperature
(21 °C) in DMSO-d6 solutions at 300 K on a Bruker Avance 300
spectrometer and referenced to the resonance signal of DMSO
(with TMS as an internal standard; chemical shifts are reported
in ppm, and coupling constants in Hz). The LC/MS analyses were
carried out on UHPLC-MS system consisting of UHPLC chromato-
graph Accela with photodiode array detector and triple quadrupole
mass spectrometer TSQ Quantum Access (both Thermo Scientific,
CA, USA), using Nucleodur Gravity C18 column at 30 °C and flow
4.1.3.1. 5-Methoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8a. Nucleoside 8a was pre-
pared from 1a (238.5 mg, 1.53 mmol), HMDS (10 mL), 1-O-acetyl-
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (847.7 mg, 1.68 mmol) and
trimethylsilyl trifluoromethanesulfonate (415 l, 2.29 mmol)
l
rate of 800
l
L/min (Macherey–Nagel, 1.8
l
m, 2.1 ꢁ 50 mm, Ger-
according to the general procedure. Yield 557.4 mg (61%), mp
78–80 °C; 1H NMR (300 MHz, DMSO-d6) d 3.15 (s, 3H) 3.90–4.04
(m, 2H) 4.58–4.80 (m, 3H) 5.94 (d, J = 4.0 Hz, 2H) 6.23 (d,
J = 3.5 Hz, 1H) 7.39–7.56 (m, 6H) 7.60–7.72 (m, 3H) 7.82 (s, 1H)
7.91 (d, J = 7.0 Hz, 2H) 7.87 (d, J = 7.0 Hz, 2H) 8.03 (d, J = 7.0 Hz,
2H) 11.61 (s, 1H). MS m/z calcd for C32H28N2O10: 600.57, found
599.10 [MꢀH]ꢀ. Anal. Calcd for C32H28N2O10 (600.57): C, 64.00;
H, 4.70; N, 4.66. Found: C, 63.7 8; H, 4.62; N, 4.43.
many). The mobile phase was (A) 0.01 M ammonium acetate in
water, and (B) acetonitrile, linearly programmed from 10% to 80%
B over 2.5 min, kept for 1.5 min. The column was pre-equilibrated
with 10% of solution B for 1 min. The purity of all compounds was
P95%. The APCI source operated at discharge current of 5 lA,
vaporizer temperature of 400 °C and capillary temperature of
200 °C. Elemental analyses were performed using an EA 1108 Ele-
mental Analyzer (Fison Instruments). Purification by column chro-
matography was carried out using silica gel.
4.1.3.2. 5-Ethoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofurano
syl)pyrimidine-2,4(1H,3H)-dione 8b. Nucleoside 8b was pre-
pared from 1b (351.4 mg, 2.07 mmol), HMDS (15 mL), 1-O-acetyl-
4.1.1. 5-[(Nonyloxy)methyl]pyrimidine-2,4(1H,3H)-dione 1i
Compound 5 (500.0 mg, 3.11 mmol) was heated in 1-nonanol
(25 mL) at 100 °C for 4 h. After cooling to rt, the precipitated solid
was collected by filtration, washed with 1-nonanol and dried. Yield
625.2 mg (75%), mp 210–214 °C; 1H NMR (300 MHz, DMSO-d6) d
0.79–0.89 (m, 3H) 1.23 (s, 12H) 1.46 (t, J = 6.0 Hz, 2H) 3.35 (t,
J = 6.5 Hz, 2H) 4.03 (s, 2H) 7.37 (d, J = 6.0 Hz, 1H) 10.83 (d,
J = 5.0 Hz, 1H) 11.11 (s, 1H). 13C NMR (75 MHz, DMSO-d6) d
14.49, 22.62, 26.20, 29.20, 29.39, 29.53, 29.70, 31.81, 64.79,
70.01, 109.80, 140.86, 151.84, 164.32. MS m/z calcd for
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (1041.8 mg, 2.07 mmol)
and trimethylsilyl trifluoromethanesulfonate (415 l, 2.29 mmol)
l
according to the general procedure. Yield 644.6 mg (69%), mp
72–75 °C; 1H NMR (300 MHz, DMSO-d6) d 1.03 (t, J = 7.0 Hz, 3H)
3.30–3.38 (m, 2H) 3.94–4.09 (m, 2H) 4.58–4.81 (m, 3H) 5.95 (d,
J = 3.0 Hz, 2H) 6.22 (d, J = 3.0 Hz, 1H) 7.40–7.56 (m, 6H) 7.60–
7.72 (m, 3H) 7.81 (s, 1H) 7.91 (d, J = 8.0 Hz, 2H) 7.87 (d,
J = 8.0 Hz, 2H) 8.03 (d, J = 7.5 Hz, 2H) 11.60 (s, 1H). MS m/z calcd
for C33H30N2O10: 614.60, found 613.18 [MꢀH]ꢀ. Anal. Calcd for
C
14H24N2O3: 268.35, found 267.10 [MꢀH]ꢀ.
C33H30N2O10 (614.60): C, 64.49; H, 4.92; N, 4.56. Found: C, 64.64;
H, 4.91; N, 4.50.
4.1.2. 5-[(Undecyloxy)methyl]pyrimidine-2,4(1H,3H)-dione 1k
Compound 5 (496.0 mg, 3.09 mmol) was heated in 1-undecanol
(25 mL) at 100 °C for 4 h. After cooling to rt, the precipitated solid
was collected by filtration, washed with the 1-undecanol and
dried. Yield 612.4 mg (67%), mp 204–206 °C; 1H NMR (300 MHz,
DMSO-d6) d 0.85 (t, J = 6.0 Hz, 3H) 1.24 (s, 16H) 1.41–1.53 (m,
2H) 3.35 (t, J = 6.0 Hz, 2H) 4.04 (s, 2H) 7.35 (br s, 1H) 10.77 (br s,
1H) 11.06 (br s, 1H). 13C NMR (75 MHz, DMSO-d6) d 14.49, 22.62,
26.20, 29.23, 29.39, 29.53, 29.56, 29.70, 31.83, 64.79, 70.02,
4.1.3.3. 5-Propoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8c. Nucleoside 8c was pre-
pared from 1c (293.8 mg, 1.60 mmol), HMDS (15 mL), 1-O-acetyl-
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (804.7 mg, 1.60 mmol) and
trimethylsilyl trifluoromethanesulfonate (320 l, 1.77 mmol)
l
according to the general procedure. Yield 437.4 mg (44%), mp
70–72 °C; 1H NMR (300 MHz, DMSO-d6) d 0.79 (t, J = 7.0 Hz, 3H)

L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
2141
1.35–1.49 (m, 2H) 3.25 (td, J = 7.0, 2.0 Hz, 2H) 4.01 (q, J = 12.0 Hz,
2H) 4.58–4.81 (m, 3H) 5.95 (d, J = 3.5 Hz, 2H) 6.23 (d, J = 3.0 Hz,
1H) 7.40–7.56 (m, 6H) 7.61–7.71 (m, 3H) 7.81 (s, 1H) 7.83–7.95
(m, 4H) 8.03 (d, J = 7.0 Hz, 2H) 11.60 (s, 1H). MS m/z calcd for
4.1.3.8. 5-Pentyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D
-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8e. Nucleoside 8e was pre-
pared from 1e (145.6 mg, 0.69 mmol), HMDS (10 mL), 1-O-acetyl-
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (346.1 mg, 0.69 mmol) and
C34H32N2O10
:
628.63, found 627.14 [MꢀH]ꢀ. Anal. Calcd for
trimethylsilyl trifluoromethanesulfonate (137 l, 0.76 mmol)
l
C34H32N2O10 (628.63): C, 64.96; H, 5.13; N, 4.46. Found: C, 64.88;
according to the general procedure. Yield 165.1 mg (37%), mp
62–64 °C; 1H NMR (300 MHz, DMSO-d6) d 0.82 (t, J = 7.0 Hz, 3H)
1.12–1.29 (m, 4H) 1.30–1.47 (m, 2H) 3.23–3.31 (m, 2H) 4.00 (q,
J = 12.0 Hz, 2H) 4.57–4.82 (m, 3H) 5.94 (d, J = 3.5 Hz, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.37–7.57 (m, 6H) 7.59–7.72 (m, 3H) 7.81 (s, 1H)
7.89 (dd, J = 12.0, 7.32 Hz, 4H) 8.03 (d, J = 7.0 Hz, 2H) 11.14 (br s,
1H). MS m/z calcd for C36H36N2O10: 656.68, found 655.14 [MꢀH]
ꢀ. Anal. Calcd for C36H36N2O10 (656.68): C, 65.84; H, 5.53; N,
4.27. Found: C, 65.98; H, 5.45; N, 4.07.
H, 5.06; N, 4.30.
4.1.3.4. 5-Isopropoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8m. Nucleoside 8m
was prepared from 1m (191.6 mg, 1.04 mmol), HMDS (15 mL),
1-O-acetyl-20,30,50-tri-O-benzoyl-b-
1.04 mmol) and trimethylsilyl trifluoromethanesulfonate (210
D
-ribofuranose
(524.8 mg,
l,
l
1.16 mmol) according to the general procedure. Yield 307.7 mg
(47%), mp 46–47 °C; 1H NMR (300 MHz, DMSO-d6) d 0.98–1.07
(m, 6H) 3.48–3.60 (m, 1H) 4.02 (q, J = 12.0 Hz, 2H) 4.58–4.81 (m,
3H) 5.95 (d, J = 4.0 Hz, 2H) 6.22 (d, J = 3.0 Hz, 1H) 7.40–7.56 (m,
6H) 7.61–7.72 (m, 3H) 7.78 (s, 1H) 7.84–7.95 (m, 4H) 8.02 (d,
4.1.3.9. 5-Hexyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8f. Nucleoside 8f was pre-
pared from 1f (341.8 mg, 1.51 mmol), HMDS (10 mL), 1-O-acetyl-
J = 7.0 Hz, 2H) 11.59 (s, 1H). MS m/z calcd for C34H32N2O10
:
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (838.3 mg, 1.66 mmol) and
628.63, found 627.11 [MꢀH]ꢀ. Anal. Calcd for C34H32N2O10
trimethylsilyl trifluoromethanesulfonate (410 l, 2.27 mmol)
l
(628.63): C, 64.96; H, 5.13; N, 4.46. Found: C, 64.78; H, 5.02; N, 4.34.
according to the general procedure. Yield 405.2 mg (40%), mp
50–51 °C; 1H NMR (300 MHz, DMSO-d6) d 0.75–0.90 (m, 3H)
1.10–1.31 (m, 6H) 1.32–1.48 (m, 2H) 3.22–3.32 (m, 2H) 4.01 (q,
J = 12.0 Hz, 2H) 4.58–4.82 (m, 3H) 5.89–6.00 (m, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.38–7.57 (m, 6H) 7.59–7.72 (m, 3H) 7.81 (s, 1H)
7.89 (dd, J = 12.0, 7.0 Hz, 4H) 8.03 (d, J = 7.0 Hz, 2H) 11.60 (s, 1H).
MS m/z calcd for C37H38N2O10: 670.71, found 669.16 [MꢀH]ꢀ. Anal.
Calcd for C37H38N2O10 (670.71): C, 66.26; H, 5.71; N, 4.18. Found: C,
66.12; H, 5.58; N, 4.29.
4.1.3.5.
5-Butoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8d. Nucleoside 8d was
prepared from 1d (166.8 mg, 0.84 mmol), HMDS (15 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (424.5 mg, 0.84 mmol)
and trimethylsilyl trifluoromethanesulfonate (170 l, 0.94 mmol)
l
according to the general procedure. Yield 185.1 mg (34%), mp
63–66 °C; 1H NMR (300 MHz, DMSO-d6) d 0.77–0.86 (m, 3H) 1.24
(sxt, J = 7.0 Hz, 2H) 1.33–1.46 (m, 2H) 3.25–3.32 (m, 2H) 4.01 (q,
J = 12.0 Hz, 2H) 4.58–4.72 (m, 2H) 4.72–4.80 (m, 1H) 5.94 (d,
J = 3.5 Hz, 2H) 6.23 (d, J = 3.0 Hz, 1H) 7.40–7.56 (m, 6H) 7.60–
7.72 (m, 3H) 7.81 (s, 1H) 7.91 (d, J = 7.5 Hz, 2H) 7.87 (d,
J = 7.0 Hz, 2H) 8.03 (d, J = 7.0 Hz, 2H) 11.59 (br s, 1H). MS m/z calcd
for C35H34N2O10: 642.65, found 641.14 [MꢀH]ꢀ. Anal. Calcd for
4.1.3.10. 5-Heptyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8g. Nucleoside 8g was
prepared from 1g (374.9 mg, 1.56 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D-ribofuranose (787.1 mg, 1.56 mmol)
and trimethylsilyl trifluoromethanesulfonate (420 l, 2.32 mmol)
l
C35H34N2O10 (642.65): C, 65.41; H, 5.33; N, 4.26. Found: C, 65.39;
according to the general procedure. Yield 526.3 mg (49%), mp 48–
50 °C; 1H NMR (300 MHz, DMSO-d6) d 0.82 (t, J = 7.0 Hz, 3H)
1.11–1.31 (m, 8H) 1.32–1.46 (m, 2H) 3.28 (td, J = 6.5, 3.0 Hz, 2H)
4.01 (q, J = 12.0 Hz, 2H) 4.57–4.81 (m, 3H) 5.94 (d, J = 4.0 Hz, 2H)
6.23 (d, J = 3.0 Hz, 1H) 7.39–7.57 (m, 6H) 7.60–7.72 (m, 3H) 7.81
(s, 1H) 7.89 (dd, J = 12.0, 7.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.60
(s, 1H). MS m/z calcd for C38H40N2O10: 684.73, found 683.14
[MꢀH]ꢀ. Anal. Calcd for C38H40N2O10 (684.73): C, 66.65; H, 5.89;
N, 4.09. Found: C, 66.48; H, 5.90; N, 4.17.
H, 5.30; N, 4.39.
4.1.3.6. 5-sec-Butoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8n. Nucleoside 8n was
prepared from 1n (303.7 mg, 1.53 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (772.9 mg, 1.53 mmol)
and trimethylsilyl trifluoromethanesulfonate (420 l, 2.32 mmol)
l
according to the general procedure. Yield 457.2 mg (46%), mp 67–
70 °C; 1H NMR (300 MHz, DMSO-d6) d 0.77 (td, J = 7.0, 2.0 Hz, 3H)
0.95–1.04 (m, 3H) 1.21–1.45 (m, 2H) 3.25–3.31 (m, 1H) 3.89–4.15
(m, 2H) 4.58–4.81 (m, 3H) 5.91–5.98 (m, 2H) 6.23 (d, J = 2.0 Hz,
1H) 7.39–7.56 (m, 6H) 7.60–7.71 (m, 3H) 7.78 (s, 1H) 7.91 (d,
J = 8.0 Hz, 2H) 7.87 (d, J = 8.0 Hz, 2H) 8.02 (d, J = 7.0 Hz, 2H) 11.59
(br s, 1H). MS m/z calcd for C35H34N2O10: 642.65, found 641.14
[MꢀH]ꢀ. Anal. Calcd for C35H34N2O10 (642.65): C, 65.41; H, 5.33;
N, 4.36. Found: C, 65.27; H, 5.26; N, 4.42.
4.1.3.11. 5-Oktyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
anosyl)pyrimidine-2,4(1H,3H)-dione 8h. Nucleoside 8h was
prepared from 1h (300.3 mg, 1.18 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D-ribofuranose (655.2 mg, 1.30 mmol)
and trimethylsilyl trifluoromethanesulfonate (320 l, 1.77 mmol)
l
according to the general procedure. Yield 379.9 mg (46%), mp
56–57 °C; 1H NMR (300 MHz, DMSO-d6) d 0.82 (t, J = 7.0 Hz, 3H)
1.11–1.30 (m, 10H) 1.32–1.47 (m, 2H) 3.23–3.32 (m, 2H) 3.92–
4.10 (m, 2H) 4.59–4.81 (m, 3H) 5.94 (d, J = 3.5 Hz, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.39–7.55 (m, 6H) 7.60–7.72 (m, 3H) 7.81 (s, 1H)
7.89 (dd, J = 12.0, 7.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.60 (s, 1H).
MS m/z calcd for C39H42N2O10: 698.76, found 697.22 [MꢀH]ꢀ. Anal.
Calcd for C39H42N2O10 (698.76): C, 67.04; H, 6.06; N, 4.01. Found: C,
67.17; H, 6.18; N, 4.21.
4.1.3.7. 5-tert-Butoxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8o. Nucleoside 8o was
prepared from 1o (186.0 mg, 0.94 mmol), HMDS (15 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (473.4 mg, 0.94 mmol)
and trimethylsilyl trifluoromethanesulfonate (190 l, 1.05 mmol)
l
according to the general procedure. Yield 248.0 mg (41%), mp
88–90 °C; 1H NMR (300 MHz, DMSO-d6) d 1.10 (s, 9H) 3.99 (d,
J = 12.0 Hz, 2H) 4.56–4.81 (m, 3H) 5.96 (br s, 2H) 6.22 (br s, 1H)
7.38–7.57 (m, 6H) 7.59–7.71 (m, 3H) 7.73 (s, 1H) 7.89 (t,
J = 8.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.57 (s, 1H). MS m/z calcd
for C35H34N2O10: 642.65, found 641.04 [MꢀH]ꢀ. Anal. Calcd for
4.1.3.12.
5-Nonyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8i. Nucleoside 8i was
prepared from 1i (505.9 mg, 1.89 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (1046.2 mg, 2.07 mmol)
and trimethylsilyl trifluoromethanesulfonate (515 l, 2.85 mmol)
according to the general procedure. Yield 623.4 mg (46%), mp
C35H34N2O10 (642.65): C, 65.41; H, 5.33; N, 4.36. Found: C, 65.55;
l
H, 5.41; N, 4.33.

2142
L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
58–60 °C; 1H NMR (300 MHz, DMSO-d6) d 0.78–0.89 (m, 3H) 1.13–
1.29 (m, 12H) 1.33–1.46 (m, 2H) 3.23–3.32 (m, 2H) 4.01 (d,
J = 11.0 Hz, 2H) 4.58–4.81 (m, 3H) 5.94 (d, J = 3.0 Hz, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.39–7.56 (m, 6H) 7.60–7.71 (m, 3H) 7.81 (s, 1H)
7.89 (dd, J = 12.0, 7.5 Hz, 4H) 8.02 (d, J = 7.5 Hz, 2H) 11.60 (s, 1H).
MS m/z calcd for C40H44N2O10: 712.78, found 711.20 [MꢀH]ꢀ. Anal.
Calcd for C40H44N2O10 (712.78): C, 67.40; H, 6.22; N, 3.93. Found: C,
67.59; H, 6.32; N, 4.05.
(300 MHz, DMSO-d6) d 0.85 (t, J = 7.0 Hz, 3H) 1.43–1.57 (m, 2H)
3.29–3.38 (m, 2H) 3.49–3.68 (m, 2H) 3.84 (q, J = 3.0 Hz, 1H)
3.92–4.12 (m, 4H) 5.05–5.13 (m, 2H) 5.39 (d, J = 5.5 Hz, 1H) 5.79
(d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s, 1H). 13C NMR (75 MHz,
DMSO-d6) d 10.46, 22.28, 60.77, 64.35, 69.81, 71.17, 73.43, 84.74,
87.54, 110.61, 138.94, 150.53, 162.57. MS m/z calcd for
C
13H20N2O7: 316.31, found 315.05 [MꢀH]ꢀ.
4.1.4.2. 5-Isopropoxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
4.1.3.13. 5-Decyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribofur-
2,4(1H,3H)-dione 2m. Nucleoside 2m was prepared from 8m
(195.1 mg, 0.27 mmol) and MeOH–NH3 solution (10 mL) according
to the general procedure. Yield 91.8 mg (94%), mp 113–115 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.09 (d, J = 6.0 Hz, 6H) 3.49–3.68 (m,
3H) 3.80–3.88 (m, 1H) 3.92–4.14 (m, 4H) 5.04–5.13 (m, 2H) 5.39
(d, J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.90 (s, 1H) 11.37 (s,
1H). 13C NMR (75 MHz, DMSO-d6) d 22.54, 22.57, 61.45, 62.43,
70.51, 71.03, 74.05, 85.39, 88.16, 111.85, 139.09, 151.18, 163.13.
MS m/z calcd for C13H20N2O7: 316.31, found 315.07 [MꢀH]ꢀ.
anosyl)pyrimidine-2,4(1H,3H)-dione 8j. Nucleoside 8j was pre-
pared from 1j (338.1 mg, 1.20 mmol), HMDS (12 mL), 1-O-acetyl-
20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (604.0 mg, 1.20 mmol) and
trimethylsilyl trifluoromethanesulfonate (325 l, 1.80 mmol)
l
according to the general procedure. Yield 425.9 mg (49%), mp
67–68 °C; 1H NMR (300 MHz, DMSO-d6) d 0.76–0.89 (m, 3H)
1.11–1.31 (m, 14H) 1.39 (t, J = 6.0 Hz, 2H) 3.23–3.32 (m, 2H)
3.93–4.08 (m, 2H) 4.59–4.81 (m, 3H) 5.94 (d, J = 3.5 Hz, 2H) 6.23
(d, J = 3.0 Hz, 1H) 7.38–7.57 (m, 6H) 7.60–7.72 (m, 3H) 7.81 (s,
1H) 7.89 (dd, J = 12.5, 7.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.60 (s,
1H). MS m/z calcd for C41H46N2O10: 726.81, found 725.21 [MꢀH]
ꢀ. Anal. Calcd for C41H46N2O10 (726.81): C, 67.75; H, 6.38; N,
3.85. Found: C, 67.86; H, 6.53; N, 3.81.
4.1.4.3. 5-Butoxymethyl-1-(b-D-ribofuranosyl)pyrimidine-2,4(1
H,3H)-dione 2d. Nucleoside 2d was prepared from 8d (157.7 mg,
0.25 mmol) and MeOH–NH3 solution (10 mL) according to the gen-
eral procedure. Yield 73.4 mg (91%), mp 125–127 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.86 (t, J = 7.0 Hz, 3H) 1.21–1.37 (m, 2H)
1.40–1.54 (m, 2H) 3.35–3.42 (m, 2H) 3.49–3.68 (m, 2H) 3.84 (d,
J = 3.5 Hz, 1H) 3.92–4.13 (m, 4H) 5.09 (br s, 2H) 5.39 (d,
J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (br s, 1H).
13C NMR (75 MHz, DMSO-d6) d 14.32, 19.37, 31.72, 61.41, 65.01,
69.83, 70.43, 74.05, 85.38, 88.18, 111.24, 139.60, 151.16, 163.21.
MS m/z calcd for C14H22N2O7: 330.33, found 329.06 [MꢀH]ꢀ.
4.1.3.14. 5-Undecyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8k. Nucleoside 8k was
prepared from 1k (215.9 mg, 0.73 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (367.5 mg, 0.73 mmol)
and trimethylsilyl trifluoromethanesulfonate (198 l, 1.09 mmol)
l
according to the general procedure. Yield 181.5 mg (34%), mp
69–72 °C; 1H NMR (300 MHz, DMSO-d6) d 0.77–0.90 (m, 3H)
1.11–1.31 (m, 16H) 1.33–1.46 (m, 2H) 3.22–3.31 (m, 2H) 3.92–
4.08 (m, 2H) 4.56–4.81 (m, 3H) 5.94 (d, J = 3.5 Hz, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.39–7.55 (m, 6H) 7.59–7.72 (m, 3H) 7.80 (s, 1H)
7.89 (dd, J = 12.5, 7.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.60 (s, 1H).
MS m/z calcd for C42H48N2O10: 740.84, found 739.27 [MꢀH]ꢀ. Anal.
Calcd for C42H48N2O10 (740.84): C, 68.09; H, 6.53; N, 3.78. Found: C,
68.23; H, 6.65; N, 3.98.
4.1.4.4. 5-sec-Butoxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
2,4(1H,3H)-dione 2n. Nucleoside 2n was prepared from 8n
(300.2 mg, 0.47 mmol) and MeOH–NH3 solution (15 mL) according
to the general procedure. Yield 147.7 mg (96%), mp 120–121 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.82 (t, J = 7.0 Hz, 3H) 1.07 (d,
J = 6.0 Hz, 3H) 1.31–1.53 (m, 2H) 3.34–3.44 (m, 1H) 3.58 (q,
J = 12.0 Hz, 2H) 3.84 (d, J = 3.0 Hz, 1H) 3.92–4.18 (m, 4H) 5.10 (d,
J = 5.0 Hz, 2H) 5.38 (d, J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.89
(s, 1H) 11.37 (s, 1H). 13C NMR (75 MHz, DMSO-d6) d 10.08, 19.59,
29.14, 61.47, 62.70, 70.54, 74.05, 76.01, 85.39, 88.10, 111.93,
139.13, 151.19, 163.15. MS m/z calcd for C14H22N2O7: 330.33,
found 329.08 [MꢀH]ꢀ.
4.1.3.15. 5-Dodecyloxymethyl-1-(20,30,50-tri-O-benzoyl-b-
D-ribo-
furanosyl)pyrimidine-2,4(1H,3H)-dione 8l. Nucleoside 8l was
prepared from 1l (359.3 mg, 1.16 mmol), HMDS (12 mL), 1-O-acet-
yl-20,30,50-tri-O-benzoyl-b-
D
-ribofuranose (583.9 mg, 1.16 mmol)
4.1.4.5. 5-tert-Butoxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
and trimethylsilyl trifluoromethanesulfonate (315 l, 1.74 mmol)
l
2,4(1H,3H)-dione 2o. Nucleoside 2o was prepared from 8o
(277.3 mg, 0.43 mmol) and MeOH–NH3 solution (15 mL) according
to the general procedure. Yield 114.6 mg (80%), mp 79–81 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.17 (s, 9H) 3.47–3.67 (m, 2H) 3.85
(d, J = 3.0 Hz, 1H) 3.91–4.09 (m, 4H) 5.01–5.14 (m, 2H) 5.38 (d,
J = 5.5 Hz, 1H) 5.81 (d, J = 5.5 Hz, 1H) 7.84 (s, 1H) 11.34 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 27.84, 56.79, 61.58, 70.65, 73.51,
74.05, 85.45, 88.10, 112.56, 138.49, 151.18, 163.03. MS m/z calcd
for C14H22N2O7: 330.33, found 329.11 [MꢀH]ꢀ.
according to the general procedure. Yield 535.4 mg (61%), mp
77–78 °C; 1H NMR (300 MHz, DMSO-d6) d 0.83 (t, J = 6.5 Hz, 3H)
1.12–1.30 (m, 18H) 1.32–1.46 (m, 2H) 3.21–3.31 (m, 2H) 3.92–
4.09 (m, 2H) 4.58–4.81 (m, 3H) 5.94 (d, J = 4.0 Hz, 2H) 6.23 (d,
J = 3.0 Hz, 1H) 7.39–7.55 (m, 6H) 7.60–7.72 (m, 3H) 7.80 (s, 1H)
7.89 (dd, J = 12.5, 7.0 Hz, 4H) 8.02 (d, J = 7.0 Hz, 2H) 11.60 (s, 1H).
MS m/z calcd for C43H50N2O10: 754.86, found 753.56 [MꢀH]ꢀ. Anal.
Calcd for C43H50N2O10 (754.86): C, 68.32; H, 6.78; N, 3.81. Found: C,
68.46; H, 6.69; N, 3.69.
4.1.4.6.
5-Pentyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
4.1.4. General procedure for preparation of 5-alkoxymethyl-1-
2,4(1H,3H)-dione 2e. Nucleoside 2e was prepared from 8e
(152.6 mg, 0.23 mmol) and MeOH–NH3 solution (20 mL) according
to the general procedure. Yield 52.6 mg (66%), mp 122–125 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.80–0.90 (m, 3H) 1.19–1.34 (m, 4H)
1.48 (quin, J = 6.5 Hz, 2H) 3.32–3.41 (m, 2H) 3.49–3.68 (m, 2H)
3.84 (d, J = 3.5 Hz, 1H) 3.92–4.12 (m, 4H) 5.06–5.13 (m, 2H) 5.39
(d, J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s,
1H). 13C NMR (75 MHz, DMSO-d6) d 14.48, 22.48, 28.40, 29.34,
61.44, 65.01, 70.15, 70.43, 74.05, 85.39, 88.20, 111.25, 139.60,
151.16, 163.21. MS m/z calcd for C15H24N2O7: 344.36, found
343.19 [MꢀH]ꢀ.
(b-D
-ribofuranosyl)pyrimidine-2,4(1H,3H)-diones 2
Nucleosides 8 were dissolved in MeOH–NH3 solution and stirred
at room temperature for 6 days. Then the mixture was evaporated,
co-evaporated with MeOH and purified by preparative chromatog-
raphy using CHCl3–MeOH (9:0.5) to afford the nucleosides 2.
4.1.4.1. 5-Propoxymethyl-1-(b-D-ribofuranosyl)pyrimidine-2,4(1
H,3H)-dione 2c. Nucleoside 2c was prepared from 8c (390.2 mg,
0.62 mmol) and MeOH–NH3 solution (15 mL) according to the gen-
eral procedure. Yield 178.2 mg (91%), mp 134–136 °C; 1H NMR

L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
2143
4.1.4.7. 5-Hexyloxymethyl-1-(b-
D
-ribofuranosyl)pyrimidine-2,4
4.1.4.12. 5-Undecyloxymethyl-1-(b-
D
-ribofuranosyl)pyrimidine-
(1H,3H)-dione 2f. Nucleoside 2f was prepared from 8f (315.7 mg,
0.47 mmol) and MeOH–NH3 solution (15 mL) according to the gen-
eral procedure. Yield 143.4 mg (85%), mp 119–121 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.85 (t, J = 6.0 Hz, 3H) 1.17–1.34 (m, 6H)
1.41–1.55 (m, 2H) 3.35–3.41 (m, 2H) 3.48–3.70 (m, 2H) 3.84 (d,
J = 3.0 Hz, 1H) 3.92–4.13 (m, 4H) 5.05–5.14 (m, 2H) 5.39 (d,
J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 14.46, 22.61, 25.85, 29.63, 31.65,
61.42, 65.01, 70.15, 70.43, 74.07, 85.39, 88.22, 111.24, 139.63,
151.16, 163.21. MS m/z calcd for C16H26N2O7: 358.39, found
357.13 [MꢀH]ꢀ.
2,4(1H,3H)-dione 2k. Nucleoside 2k was prepared from 8k
(161.4 mg, 0.22 mmol) and MeOH–NH3 solution (10 mL) according
to the general procedure. Yield 84.5 mg (91%), mp 131–133 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.75–0.90 (m, 3H) 1.24 (s, 16H) 1.38–
1.55 (m, 2H) 3.34–3.40 (m, 2H) 3.50–3.69 (m, 2H) 3.84 (d,
J = 3.5 Hz, 1H) 3.91–4.12 (m, 4H) 5.03–5.14 (m, 2H) 5.39 (d,
J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 13.86, 22.00, 25.56, 28.63, 28.80,
28.92, 28.95, 28.95, 29.04, 31.20, 60.79, 64.39, 69.53, 69.80,
73.43, 84.75, 87.57, 110.59, 138.99, 150.53, 162.57. MS m/z calcd
for C21H36N2O7: 428.52, found 427.25 [MꢀH]ꢀ.
4.1.4.13. 5-Dodecyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
4.1.4.8. 5-Heptyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-2,4
2,4(1H,3H)-dione 2l. Nucleoside 2l was prepared from 8l
(530.1 mg, 0.70 mmol) and MeOH–NH3 solution (20 mL) according
to the general procedure. Yield 250.1 mg (81%), mp 132–133 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.79–0.90 (m, 3H) 1.23 (s, 18H) 1.40–
1.53 (m, 2H) 3.34–3.40 (m, 2H) 3.49–3.69 (m, 2H) 3.81–3.88 (m,
1H) 3.92–4.12 (m, 4H) 5.05–5.13 (m, 2H) 5.39 (d, J = 5.5 Hz, 1H)
5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) d 14.48, 22.64, 26.20, 29.25, 29.26, 29.44,
29.56, 29.57, 29.60, 29.67, 31.84, 61.42, 65.03, 70.17, 70.43,
74.07, 85.39, 88.23, 111.24, 139.63, 151.16, 163.21. MS m/z calcd
for C22H38N2O7: 442.55, found 441.25 [MꢀH]ꢀ.
(1H,3H)-dione 2g. Nucleoside 2g was prepared from 8g
(504.0 mg, 0.74 mmol) and MeOH–NH3 solution (20 mL) according
to the general procedure. Yield 236.7 mg (86%), mp 120–122 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.78–0.91 (m, 3H) 1.24 (br s, 8H)
1.41–1.54 (m, 2H) 3.35–3.41 (m, 2H) 3.49–3.69 (m, 2H) 3.84 (d,
J = 3.5 Hz, 1H) 3.90–4.13 (m, 4H) 5.04–5.14 (m, 2H) 5.39 (d,
J = 5.5 Hz, 1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.39 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 13.86, 21.96, 25.50, 28.43, 29.04,
31.16, 60.79, 64.39, 69.50, 69.80, 73.43, 84.75, 87.57, 110.59,
139.01, 150.53, 162.57. MS m/z calcd for C17H28N2O7: 372.41,
found 371.13 [MꢀH]ꢀ.
4.2. Biological activity
4.1.4.9. 5-Octyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-2,4
(1H,3H)-dione 2h. Nucleoside 2h was prepared from 8h
(343.1 mg, 0.49 mmol) and MeOH–NH3 solution (15 mL) according
to the general procedure. Yield 188.6 mg (99%), mp 124–125 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.78–0.91 (m, 3H) 1.24 (s, 10H) 1.47
(br s, 2H) 3.35–3.41 (m, 2H) 3.48–3.68 (m, 2H) 3.84 (d, J = 3.0 Hz,
1H) 3.91–4.13 (m, 4H) 5.09 (d, J = 3.0 Hz, 2H) 5.39 (d, J = 5.5 Hz,
1H) 5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.38 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) d 14.48, 22.62, 26.20, 29.22, 29.38, 29.67,
31.80, 61.42, 65.01, 70.15, 70.43, 74.07, 85.39, 88.22, 111.24,
139.63, 151.16, 163.21. MS m/z calcd for C18H30N2O7: 386.44,
found 385.16 [MꢀH]ꢀ.
4.2.1. Cell lines
All cells were purchased from the American Tissue Culture Col-
lection (ATCC), unless otherwise indicated: the CEM line are highly
chemosensitive T-lymphoblastic leukaemia cells, K562 cells were
derived from patient with acute myeloid leukaemia with bcr-abl
translocation, A549 line is lung adenocarcinoma, HCT116 is colo-
rectal tumour cell line and its p53 gene knock-down counterpart
(HCT116p53-, Horizon Discovery, UK) is a model of human cancers
with p53 mutation frequently associated with poor prognosis. The
daunorubicin-resistant subline of CEM cells (CEM-DNR bulk) and
paclitaxel resistant subline K562-tax were selected in our labora-
tory by the cultivation of maternal cell lines in increasing concen-
trations of daunorubicine or paclitaxel, respectively.17 The CEM-
DNR bulk cells overexpress MRP-1 protein, while K562-tax cells
overexpress P-glycoprotein, both proteins belong to family of
ABC transporters and are involved in primary and/or acquired mul-
tidrug resistance phenomenon.17 The cells were maintained in
Nunc/Corning 80 cm2 plastic tissue culture flasks and cultured in
cell culture medium (DMEM/RPMI 1640 with 5 g/L glucose,
4.1.4.10. 5-Nonyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-
2,4(1H,3H)-dione 2i. Nucleoside 2i was prepared from 8i
(578.6 mg, 0.81 mmol) and MeOH–NH3 solution (20 mL) according
to the general procedure. Yield 251.5 mg (77%), mp 125–127 °C; 1H
NMR (300 MHz, DMSO-d6) d 0.79–0.91 (m, 3H) 1.24 (s, 12H) 1.47
(t, J = 6.0 Hz, 2H) 3.36–3.42 (m, 2H) 3.49–3.69 (m, 2H) 3.84 (d,
J = 3.5 Hz, 1H) 3.91–4.12 (m, 4H) 5.09 (br s, 2H) 5.39 (d,
J = 5.0 Hz, 1H) 5.79 (d, J = 5.0 Hz, 1H) 7.93 (s, 1H) 11.38 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d 14.51, 22.64, 26.20, 29.23, 29.44,
29.54, 29.67, 31.83, 61.42, 65.01, 70.15, 70.43, 74.07, 85.39,
88.20, 111.22, 139.65, 151.16, 163.22. MS m/z calcd for
2 mM glutamine, 100 U/mL penicillin, 100
10% foetal calf serum, and NaHCO3).
lg/mL streptomycin,
4.2.2. Cytotoxic MTT assay18
Cell suspensions were prepared and diluted according to the
particular cell type and the expected target cell density (2500–
30,000 cells/well based on cell growth characteristics). Cells were
added by pipette (80
were allowed a pre-incubation period of 24 h at 37 °C and 5% CO2
for stabilization. Four-fold dilutions, in 20 L aliquots, of the in-
tended test concentration were added to the microtiter plate wells
at time zero. All test compound concentrations were examined in
duplicate. Incubation of the cells with the test compounds lasted
for 72 h at 37 °C, in a 5% CO2 atmosphere at 100% humidity. At
the end of the incubation period, the cells were assayed using
C
19H32N2O7: 400.47, found 399.19 [MꢀH]ꢀ.
4.1.4.11. 5-Decyloxymethyl-1-(b-D-ribofuranosyl)pyrimidine-2,4
lL) into 96-well microtiter plates. Inoculates
(1H,3H)-dione 2j. Nucleoside 2j was prepared from 8j (347.3 mg,
0.48 mmol) and MeOH–NH3 solution (15 mL) according to the gen-
eral procedure. Yield 175.3 mg (89%), mp 129–131 °C; 1H NMR
(300 MHz, DMSO-d6) d 0.80–0.90 (m, 3H) 1.24 (s, 14H) 1.41–1.54
(m, 2H) 3.34–3.39 (m, 2H) 3.48–3.69 (m, 2H) 3.84 (d, J = 3.5 Hz,
1H) 3.91–4.12 (m, 4H) 5.04–5.13 (m, 2H) 5.38 (d, J = 5.5 Hz, 1H)
5.79 (d, J = 5.5 Hz, 1H) 7.93 (s, 1H) 11.38 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) d 14.49, 22.64, 26.20, 29.25, 29.44, 29.53,
29.58, 29.67, 31.84, 61.42, 65.03, 70.17, 70.43, 74.07, 85.39,
88.23, 111.24, 139.63, 151.16, 163.21. MS m/z calcd for
l
MTT. Aliquots (10 lL) of the MTT stock solution were pipetted into
each well and incubated for a further 1–4 h. After this incubation
period the formazan produced was dissolved by the addition of
100 lL/well of 10% aq SDS (pH 5.5), followed by a further incuba-
C
20H34N2O7: 414.49, found 413.18 [MꢀH]ꢀ.

2144
L. Brulíková et al. / Carbohydrate Research 346 (2011) 2136–2144
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Acknowledgments
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2001, 28, 249–257.
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This study was supported by grants from the Ministry of
Schools, Youth and Education of the Czech Republic (MSM
6198959216, MSM 6198959223 and LC07107), EEA/Norway Finan-
cial Mechanisms (CZ0099) and for project CZ.1.07/2.3.00/20.0009
coming from European Social Fund. The infrastructure part of this
project (Institute of Molecular and Translational Medicine) was
supported from the Operational Program Research and Develop-
ment for Innovations (project CZ.1.05/2.1.00/01.0030).
ˇ
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Med. Chem. Lett. 2007, 17, 6647–6650.
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