Synthesis of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uridines and study of their cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2010.05.003

A series of uridine analogues modified at the 5-position with the 5-[alkoxy-(4-nitrophenyl)-methyl] moiety was synthesized. Nucleosides were formed as a mixture of two diastereoisomers, which were separated and tested for their cytotoxic activity in vitro against different cancer cell lines and for antimicrobial activity. Relationships between structure and the above mentioned activities were studied. The cytotoxic activity was slightly increased in some cases by transformation of bases to nucleosides. Depending on the length of the alkyl chain increased cytotoxic and antimicrobial activity were noted. The cytotoxic activity of the nucleosides was not due to cell cycle alterations, DNA and/or RNA synthesis. The synthesis, cytotoxic activity, DNA/RNA synthesis inhibition and apoptosis induction of target compounds in their both diastereoisomeric forms were studied.

Full text of DOI:10.1016/j.ejmech.2010.05.003

European Journal of Medicinal Chemistry 45 (2010) 3588e3594
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uridines and study of their
cytotoxic activity
,
1
b,
Lucie Brulíková a , Petr Dzubák 1, Marián Hajdúch b, Lenka Lachnitová b, Madhusudhan Kollareddy b,
ꢀ
c
,
ꢀ a
*
ꢀc
ꢀ
Milan Kolár , Katerina Bogdanová , Jan Hlavác
Department of Organic Chemistry, Faculty of Science, Palacký University, Tr. Svobody 8, 771 46 Olomouc, Czech Republic
Laboratory of Experimental Medicine, Department of Pediatrics and Oncology, Faculty of Medicine, Palacký University and Faculty Hospital in Olomouc, Puskinova 6, 775 20
Olomouc, Czech Republic
Department of Microbiology, Faculty of Medicine and Dentistry, Palacký University, Hnevotínská 3, 775 15, Olomouc, Czech Republic
a
ꢀ
b
ꢀ
c
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of uridine analogues modified at the 5-position with the 5-[alkoxy-(4-nitrophenyl)-methyl]
moiety was synthesized. Nucleosides were formed as a mixture of two diastereoisomers, which were
separated and tested for their cytotoxic activity in vitro against different cancer cell lines and for anti-
microbial activity. Relationships between structure and the above mentioned activities were studied. The
cytotoxic activity was slightly increased in some cases by transformation of bases to nucleosides.
Depending on the length of the alkyl chain increased cytotoxic and antimicrobial activity were noted. The
cytotoxic activity of the nucleosides was not due to cell cycle alterations, DNA and/or RNA synthesis.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 12 September 2009
Received in revised form
5 May 2010
Accepted 6 May 2010
Available online 12 May 2010
Keywords:
Uracils
Ribonucleosides
Cytotoxic activity
Antimicrobial activity
1. Introduction
Our recent interest was centered on the 5 position of pyrimidine
nucleobases as potential anticancer agents. We modified the above
Modern synthesis in the field of drug discovery is focused on
several types of derivatives. Nucleoside analogues, as potential
inhibitors of nucleic acid metabolism, play a significant role in
this area. In this context, we could mention as examples, the
reverse transcriptase inhibitors as potential AIDS therapies [1,2]
as well as inhibitors of thymidylate synthase used for the treat-
ment of leukemias or solid tumors [3,4]. Since the discovery of
the anticancer agent 5-fluorouracil, which has been used against
cancer for about 40 years, or drugs possessing antiviral activity,
such as 5-ethyl-20-deoxyuridine, investigation of the chemistry of
pyrimidine nucleosides modified at the 5 position has grown
extensively. The synthesis of alkoxy-uracil derivatives [5e13] and
their use as sensitive and valuable markers for studies on DNA
oxidation damage, has been described [14]. Also, another
5-modified uracil e 5,50-(4-nitrophenyl)-methyl-bis-1H-pyrimi-
dine 2,4-dione - has been shown to possess significant biological
properties and is patented in the class of anti-ictogenic or anti-
epileptogenic agents [15].
mentioned alkoxy-uracils by introducing a nitro-phenyl moiety and
reported the synthesis of 5-[alkoxy-(4-nitro-phenyl)-methyl]-
uracils 3ae3o (Scheme 1) and studied their in vitro anticancer
activity against five tumor cell lines under in-vitro conditions [16].
2. Results and discussion
2.1. Chemistry
Despite finding an interesting relationship between structure
and cytotoxic activity, the values of IC50 remained beyond micro-
molar concentrations. Consequently, we decided to convert these
structures into the ribonucleosides in order to improve solubility
and increase their possibility to interact with the enzymes
responsible for nucleic acids transformation and/or biosynthesis.
Now we have selected derivatives 3fe3i with the highest
activity and transformed them into the corresponding nucleosides.
The Vorbrüggen method [17] e as the most widely used reaction for
the preparation of ribonucleosides - was used. This reaction utilizes
silylated nucleobases and strong Lewis acids.
* Corresponding author. Tel.: þ420 585 634 405; fax: þ420 585 634 467.
The synthesis was initiated by silylation of the starting
compounds 3fe3i (Scheme 2). Silylated uracils reacted with
ꢀ
E-mail address: hlavac@orgchem.upol.cz (J. Hlavác).
Both authors contributed equally to the work.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.003

L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
3589
Treatment of ribonucleosides 5fe5i and 6fe6i with methanolic
ammonia at room temperature afforded the nucleosides 7fe7i and
8fe8i.
NO2
NO2
O
NH
b
a
O
O
R
2.2. Cytotoxic activity
N
H
O
O
NH
Cl
NH
The prepared nucleosides 7fe7i and 8fe8i were tested under
in-vitro conditions for their cytotoxic activity against cancer cell
lines including drug sensitive (CEM and K-562) as well as drug
resistant (CEM-DNR-B and K-562 TAX) cell lines and A549 cells as
representative of solid tumors (Table 1) [18,19]. Cytotoxic activity
was analyzed in comparison to purine based anticancer agents
6-thioguanine and fludarabine. Although pyrimidine derivative
5-fluorouracil is structurally more related to our compounds, it was
not used as a comparative agent due to low activity in hemato-
poietic tumors, Figs. 1e5 show the activity-chain length depen-
dence. Compounds with longer alkyl chains exhibited a trend to
higher cytotoxicity in all tested cell lines. Nucleosides are not
always more potent than the free bases. The most active
compounds 7h, 7i and 8h, 8i were tested for their effect on the cell
cycle alterations including pH3Ser10 positive mitotic cells, apoptosis,
DNA and RNA synthesis inhibition (Table 2) in treated versus
control CEM T-lymphoblastic leukemia cells. Analyses were per-
formed at equiactive concentrations corresponding to 1ꢀ IC50 or
5ꢀ IC50 in a 24 h treatment interval. However, in these assays we
did not observe significant changes in cell distribution during the
cell cycle or differences in percentage of mitotic cells based on the
monitoring of pH3Ser10 positivity. All tested compounds provided
massive induction of apoptosis at 5ꢀ IC50 which was accompanied
by complete inhibition of DNA and RNA synthesis. However, at
a concentration of 1ꢀ IC50 we observed only small differences in
DNA/RNA synthetic activity suggesting that nucleic acid biosyn-
thesis is not a primary target of our compounds. On the contrary,
control agents, 6-thioguanine and fludarabine, inhibited DNA and/
or RNA synthesis even at low cytotoxic concentrations (Table 2).
These data correspond to our previous study on the free bases of
tested compounds [16].
1
N
H
O
N
H
O
2
3
a) 4-nitrobenzaldehyde, conc. HCl, reflux, 4h
b) alcohol, reflux, 4h
a) R = -CH3
h) R = -(CH2)7CH3
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
n) R = -s-But
o) R = -t-But
Scheme 1. Preparation of 5-[alkoxy-(4-nitro)-methyl]-uracils 3ae3o [16].
protected sugar in the presence of 1.1 equivalents of TMSOTf at
room temperature to afford benzoylated ribonucleosides 4fe4i that
were formed as a mixture of two diastereoisomers.
The separation of diastereoisomers was not simple due to the
similar properties of both isomers. The isomeric structures from
each mixture 4fe4i, however, had almost the same Rf and it was
therefore very difficult to separate them in amounts sufficient for
all studies and analyses. This separation was very sensitive to the
presence of water and size of column. Furthermore, a carefully
chosen gradient of the mobile phase was one of the most influen-
cial factors. Nevertheless, we have found an acceptable mobile
phase and conditions for separation by silica gel column chroma-
tography. Two diastereoisomers 5fe5i and 6fe6i from each
mixture 4fe4i were isolated using methanol in chloroform (0e5%).
NO2
NO2
NO2
NO2
separation of
diastereoisomers
O
O
N
O
N
b
a
R
R
R
O
NH
O
NH
O
O
NH
*
R
O
O
O
NH
N
O
HO
BzO
BzO
O
OH OH
O
OBz OBz
5f) R = -(CH2)5-CH3 ; [
5g) R = -(CH2)6-CH3 ; [
5h) R = -(CH2)7-CH3 ; [
5i) R = -(CH2)8-CH3; [
6f) R = -(CH2)5-CH3 ; [
6g) R = -(CH2)6-CH3 ; [
6h) R = -(CH2)7-CH3 ; [
6i) R = -(CH2)8-CH3 ; [
N
H
O
O
3
OBz OBz
4
(+)-isomers
(-)-isomers
7
8
α
]
25 +151.9
25 +32.7
D
f) R = -(CH2)5-CH3
g) R = -(CH2)6-CH3
h) R = -(CH2)7-CH3
i) R = -(CH2)8-CH3
α
α
]
]
D
]
25 +38.5
D
f) R = -(CH2)5-CH3
g) R = -(CH2)6-CH3
h) R = -(CH2)7-CH3
i) R = -(CH2)8-CH3
α
25 +90.2
D
α
]
25 +213.9
D
α
α
]
]
25 +48.6
25 +56.8
D
]
D
α
25 +56.4
D
a) hexamethyldisilazane, (NH4)2SO4, 1-O-acetyl-2,3,5-tri-O-benzoyl-
anhydrous 1,2-dichloroethane, RT, 2 days;
b) MeOH/NH3, RT, 6 days
β-D-ribofuranose, trimethylsilyl trifluoro-methanesulfonate,
Scheme 2. Preparation of nucleosides 7fe7i and 8fe8i.

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L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
Table 1
Cytotoxic activity of standard anticancer agents and derivatives 7fe7i and 8fe8i on human malignant cell lines of different tissue origin and drug resistance profile.
Compounds
R
A 549
CEM
IC50
(
m
M)
K 562
K562-TAX
CEM-DNR-B
6-thioguanine
fludarabine
7f
7g
7h
7i
2.82 ꢂ 1.2
2.98 ꢂ 0.8
19.49 ꢂ 0.9
39.8 ꢂ 1.9
12.8 ꢂ 3.5
11.4 ꢂ 1.7
7.9 ꢂ 1.4
9.47 ꢂ 1.2
1.01 ꢂ 0.34
131.0 ꢂ 7.1
103.7 ꢂ 9.8
34.6 ꢂ 2.4
32.3 ꢂ 3.0
145.8 ꢂ 12.6
95.6 ꢂ 12.1
36.6 ꢂ 3.1
33.8 ꢂ 2.8
1.54 ꢂ 0.07
267 ꢂ 22.5
58.4 ꢂ 3.1
33.6 ꢂ 1.8
15.1 ꢂ 2.8
10.9 ꢂ 1.4
64.5 ꢂ 12.3
36.7 ꢂ 2.2
24.7 ꢂ 7.7
13.0 ꢂ 1.1
3.9 ꢂ 0.56
0.26 ꢂ 0.15
141.4 ꢂ 14.3
42.8 ꢂ 3.6
33.1 ꢂ 3.4
14.6 ꢂ 3.5
136.9 ꢂ 14.3
52.3 ꢂ 4.4
34.4 ꢂ 2.7
23.9 ꢂ 5.8
47.44 ꢂ 9.0
121.3 ꢂ 23.4
39.8 ꢂ 13.5
38.7 ꢂ 11.0
23.1 ꢂ 11.0
156.0 ꢂ 22.0
69.9 ꢂ 35.8
39.7 ꢂ 10.3
29.1 ꢂ 11.1
hexyl
heptyl
oktyl
nonyl
hexyl
heptyl
oktyl
8f
45.5 ꢂ 16.5
29.0 ꢂ 4.9
16.9 ꢂ 5.1
10.0 ꢂ 1.0
8g
8h
8i
nonyl
2.3. Antimicrobial activity
4. Experimental section
The compounds were also tested for their antimicrobial activity
(see Table 3) against standard reference gram-positive and gram-
negative bacterial strains (Enterococcus faecalis CCM 4224, Staphy-
lococcus aureus CCM 3953, Escherichia coli CCM 3954 and Pseudo-
monas aeruginosa CCM 3955) from the Czech Collection of
Microorganisms (CCM, Faculty of Science, Masaryk University
Brno), and against gram-positive and gram-negative bacteria
obtained from clinical material of patients treated at University
Hospital in Olomouc (methicillin resistant S. aureus e MRSA,
Staphylococcus haemolyticus, E. coli and P. aeruginosa) with resis-
tance to fluoroquinolones used in clinical practice. Only the octyl
and nonyl derivatives 7h, 8h and 7i, 8i showed slight activity
against E. faecalis CCM 4224, S. aureus CCM 3953, S. aureus MRSA
and S. haemolyticus.
4.1. Chemistry
Melting points were determined on a Boetius stage and are
uncorrected. 1H NMR spectra were measured in DMSO-d6 at 300 K
on a Bruker Avance 300 spectrometer (300 MHz with TMS as an
internal standard; chemical shifts are reported in ppm, and
coupling constants in Hz). Mass spectrometric experiments were
performed using a triple quadrupole mass spectrometer TSQ
Quantum Access and chromatographic analysis were performed
using ultra-high pressure liquid chromatograph Accela (both from
Thermo Scientific, San Jose, CA, USA). HPLC experiments were
performed using Dionex liquid chromatograph (P 680 HPLC Pump,
PDA-100 Photodiode Array Detector). Preparative chromatography
was performed with using of Sepacore chromatography system
(Büchi). Optical rotations were recorded at the Sodium D line with
a polarimeter at room temperature.
3. Conclusion
4.1.1. 20,30,50-tri-O-benzoyl-
b-D-ribofuranosyl-5-[hexyl-(4-nitro-
phenyl)-methyl]-pyrimidine-2,4-dione (4f), (5f), (6f)
5-[Hexyl-(4-nitro-phenyl)-methyl]-1H-pyrimidine-2,4-dione
(3f) (600 mg, 1.7273 mmol) was heated in hexamethyldisilazane
(15 ml) at 140 ꢁC with (NH4)2SO4 (approximately 5 mg) for 8 h.
After that it was evaporated and residue was dissolved in anhy-
drous 1,2-dichloroethane (20 ml). To this solution, 1-O-acetyl-
In conclusion, insertion of a ribose moiety into nucleosides
derived from 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracil did not
bring about a significant increase of cytotoxic activity against
cancer lines in comparison to the analogous free bases. The activity
of these nucleosides was higher in drug sensitive than in drug
resistant cancer lines and was independent of the chirality of the
molecule. In contrast to the free bases, the nucleosides exhibited
only weak activity against several bacterial strains suggesting
specific target(s) in eukaryotic cells.
2,3,5-tri-O-benzoyl-
b-
D-ribofuranose (871.4 mg, 1.7273 mmol) and
trimethylsilyl trifluoro-methanesulfonate (345
ml, 1.9061 mmol)
were added. This mixture was stirred at room temperature for 2
days, washed with water (20 ml) and ethyl acetate (60 ml), dried
over sodium sulphate, filtered and evaporated to dryness. Yield of
Because the activity increases with chain length in both anti-
cancer as well as antimicrobial activity, derivatives with longer
chains might be promising for future study of their activity and
mechanism of action.
Fig. 1. Cytotoxic activity of compounds 3fe3i, 6fe6i and 7fe7i as a function of chain
Fig. 2. Cytotoxic activity of compounds 3fe3i, 7fe7i and 8fe8i as a function of chain
length in the CEM cell line.
length in the K562 cell line.

L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
3591
Fig. 5. Cytotoxic activity of compounds 3fe3i, 7fe7i and 8fe8i as a function of chain
length in CEM-DNR-B cell line.
Fig. 3. Cytotoxic activity of compounds 3fe3i, 7fe7i and 8fe8i as a function of chain
length in the K562-TAX cell line.
crude product (4f) (mixture of diastereoisomers) 852.3 mg (62%).
Two diastereoisomers (5f) and (6f) were isolated in this order from
the mixture by silica gel column chromatography using methanol
in chloroform (0e5%).
anhydrous 1,2-dichloroethane (20 ml). To this solution, 1-O-acetyl-
2,3,5-tri-O-benzoyl-
b
-D-ribofuranose (837.6 mg, 1.6603 mmol) and
trimethylsilyl trifluoro-methanesulfonate (331
ml, 1.8288 mmol)
were added. This mixture was stirred at room temperature for 2
days, washed with water (20 ml) and ethyl acetate (60 ml), dried
over sodium sulphate, filtered and evaporated to dryness. Yield of
crude product (4g) (mixture of diastereoisomers) 954.5 mg (71%).
Two diastereoisomers (5g) and (6g) were isolated in this order from
the mixture by silica gel column chromatography using methanol
in chloroform (0e5%).
4.1.1.1. Isomer (5f). Yield 189.3 mg (14%), m.p. 75e77 ꢁC,
[
a
]
25 þ 151.9 (c 0.027, CHCl3); 1H NMR (DMSO-d6):
d 0.76e0.81 (m,
D
3H, CH3); 1.12e1.26 (m, 6H, CH2); 1.37e1.47 (m, 2H, CH2);
3.23e3.44 (m, 2H, CH2); 4.63e4.65 (m, 2H, H-50); 4.77e4.81 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.94e6.02 (m, 2H, H-20, H-30); 6.25 (d, 1H, H-
10, J ¼ 4.2 Hz); 7.41e7.51 (m, 6H, Ph); 7.57 (d, 2H, Ph, J ¼ 8.7 Hz);
7.61e7.69 (m, 3H, Ph); 7.82 (s, 1H, Ph); 7.86 (d, 2H, Ph, J ¼ 7.2 Hz);
7.93 (d, 2H, Ph, J ¼ 7.2 Hz); 8.02 (d, 2H, Ph, J ¼ 7.2 Hz); 8.15 (d, 2H,
4.1.2.1. Isomer (5g). Yield 372.7 mg (28%), m.p. 72e74 ꢁC,
25
[a
]
þ 32.7 (c 0.11, CHCl3); 1H NMR (DMSO-d6):
d 0.77e0.82 (m,
D
Ph, J ¼ 8.7 Hz); 11.63 (s, 1H, NH). MS m/z Calc. for C43H41N3O12
:
3H, CH3); 1.20e1.23 (m, 8H, CH2); 1.38e1.46 (m, 2H, CH2);
3.25e3.44 (m, 2H, CH2); 4.64e4.65 (m, 2H, H-50); 4.77e4.81 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.94e6.01 (m, 2H, H-20, H-30); 6.25 (d, 1H, H-
10, J ¼ 4.5 Hz); 7.41e7.51 (m, 6H, Ph); 7.56e7.59 (m, 2H, Ph);
7.62e7.69 (m, 3H, Ph); 7.82e7.87 (m, 3H, Ph); 7.91e7.94 (m, 2H, Ph);
8.00e8.03 (m, 2H, Ph); 8.14 (d, 2H, Ph, J ¼ 9.0 Hz); 11.64 (s, 1H, NH).
MS m/z Calc. for C44H43N3O12: 805.85, found 804.25 [M ꢃ H]ꢃ.
791.82, found 790.25 [M ꢃ H]ꢃ.
4.1.1.2. Isomer (6f). Yield 167.2 mg (12%), m.p. 69e72 ꢁC,
25
[
a]
þ 213.9 (c 0.018, CHCl3); 1H NMR (DMSO-d6):
d 0.81 (t, 3H,
D
CH3, J ¼ 7.2 Hz); 1.19e1.26 (m, 6H, CH2); 1.42e1.50 (m, 2H, CH2);
3.23e3.45 (m, 2H, CH2); 4.62e4.68 (m, 2H, H-50); 4.73e4.78 (m, 1H,
H-40); 5.33 (s, 1H, CH); 5.95e6.02 (m, 2H, H-20, H-30); 6.21 (d, 1H, H-
10, J ¼ 3.6 Hz); 7.42e7.53 (m, 6H, Ph); 7.58e7.68 (m, 5H, Ph); 7.79 (s,
1H, Ph); 7.88e7.91 (m, 4H, Ph); 8.02 (d, 2H, Ph, J ¼ 6.9 Hz); 8.15 (d,
4.1.2.2. Isomer (6g). Yield 361.5 mg (27%), m.p. 65e66 ꢁC,
[
a
]
25 þ 48.6 (c 0.074, CHCl3); 1H NMR (DMSO-d6):
d 0.78e0.82 (m,
D
2H, Ph, J ¼ 8.7 Hz); 11.64 (s, 1H, NH). MS m/z Calc. for C43H41N3O12
:
3H, CH3); 1.19e1.26 (m, 8H, CH2); 1.42e1.52 (m, 2H, CH2);
3.25e3.45 (m, 2H, CH2); 4.62e4.65 (m, 2H, H-50); 4.73e4.78 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.95e6.02 (m, 2H, H-20, H-30); 6.21 (d, 1H, H-
10, J ¼ 3.9 Hz); 7.42e7.52 (m, 6H, Ph); 7.58e7.68 (m, 5H, Ph); 7.80 (s,
1H, Ph); 7.87e7.91 (m, 4H, Ph); 8.00e8.03 (m, 2H, Ph); 8.15 (d, 2H,
791.82, found 790.26 [M ꢃ H]ꢃ.
4.1.2. 20,30,50-tri-O-benzoyl-
b-D-ribofuranosyl-5-[heptyl-(4-nitro-
phenyl)-methyl]-pyrimidine-2,4-dione (4g), (5g), (6g)
5-[Heptyl-(4-nitro-phenyl)-methyl]-1H-pyrimidine-2,4-dione
Ph, J ¼ 8.7 Hz); 11.64 (s, 1H, NH). MS m/z Calc. for C44H43N3O12
:
(3g) (600 mg, 1.6602 mmol) was heated in hexamethyldisilazane
(15 ml) at 140 ꢁC with (NH4)2SO4 (approximately 5 mg) for 8 h.
After that it was evaporated and residue was dissolved in
805.85, found 804.25 [M ꢃ H]ꢃ.
4.1.3. 20,30,50-tri-O-benzoyl-
b-D-ribofuranosyl-5-[oktyl-(4-nitro-
phenyl)-methyl]-pyrimidine-2,4-dione (4h), (5h), (6h)
5-[Oktyl-(4-nitro-phenyl)-methyl]-1H-pyrimidine-2,4-dione
(3h) (600 mg, 1.5982 mmol) was heated in hexamethyldisilazane
(15 ml) at 140 ꢁC with (NH4)2SO4 (approximately 5 mg) for 8 h.
After that it was evaporated and residue was dissolved in anhy-
drous 1,2-dichloroethane (20 ml). To this solution,1-O-acetyl-2,3,5-
tri-O-benzoyl-
b-
D-ribofuranose (806.3 mg, 1.5982 mmol) and tri-
methylsilyl trifluoro-methanesulfonate (319
ml, 1.7625 mmol) were
added. This mixture was stirred at room temperature for 2 days,
washed with water (20 ml) and ethyl acetate (60 ml), dried over
sodium sulphate, filtered and evaporated to dryness. Yield of crude
product (4h) (mixture of diastereoisomers) 842.3 mg (67%). Two
diastereoisomers (5h) and (6h) were isolated in this order from the
mixture by silica gel column chromatography using methanol in
chloroform (0e5%).
Fig. 4. Cytotoxic activity of compounds 3fe3i, 7fe7i and 8fe8i as a function of chain
length in the A549 cell line.

3592
L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
Table 2
Summary of conventional cell cycle, apoptosis, RNA/DNA e BrU/BrDU analysis of CEM leukemia cell line treated with standard anticancer drugs or the most potent compounds
7h, 7i and 8h, 8i. Data are expressed as a percentage of positive cells in the total cell population.
Compounds
Apoptosis (%)
G0/G1 (%)
S (%)
G2/M (%)
BrUþ (%)
BrDUþ (%)
pH3Ser10þ (%)
Control
3
44.87
43.42
43.58
39.66
42.11
42.36
ND
40.95
ND
40.02
ND
44.37
49.39
46.47
40.84
52.94
47.81
ND
47.81
ND
48.56
ND
10.76
7.19
9.95
19.5
4.95
9.83
ND
11.25
ND
11.33
ND
47.59
45.84
48.93
7.62
8.92
58.1
1.44
60.22
0.8
56.18
1.61
57.26
1.9
58.56
35.32
19.82
14.12
10.37
46.03
6.01
52.17
2.95
48.73
6.67
1.35
1.21
0.43
0.28
0.01
2.05
0.03
1.66
0.25
1.68
0.01
1.74
0.04
6-thioguanine 1ꢀ IC50
6-thioguanine 5ꢀ IC50
fludarabine 1ꢀ IC50
fludarabine 5ꢀ IC50
7h 1ꢀ IC50
11.27
18.69
62.1
74.84
7
76
4
83
5
7h 5ꢀ IC50
7i 1ꢀ IC50
7i 5ꢀ IC50
8h 1ꢀ IC50
8h 5ꢀ IC50
71
4
83
8i 1ꢀ IC50
43.38
ND
47.79
ND
8.83
ND
47.38
0.95
8i 5ꢀ IC50
4.1.3.1. Isomer (5h). Yield 260.0 mg (21%), m.p. 69e70 ꢁC,
4.1.4.1. Isomer (5i). Yield 207.7 mg (16%), m.p. 62e63 ꢁC,
[
a]
25 þ 38.5 (c 0.091, CHCl3); 1H NMR (DMSO-d6):
d
0.81 (t, 3H, CH3,
[a
]
25 þ 90.2 (c 0.041, CHCl3); 1H NMR (DMSO-d6):
d 0.82 (t, 3H, CH3,
D
D
J ¼ 7.2 Hz); 1.16e1.23 (m, 10H, CH2); 1.37e1.46 (m, 2H, CH2);
3.22e3.44 (m, 2H, CH2); 4.64e4.65 (m, 2H, H-50); 4.76e4.81 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.94e6.01 (m, 2H, H-20, H-30); 6.25 (d, 1H, H-
10, J ¼ 4.5 Hz); 7.41e7.51 (m, 6H, Ph); 7.57 (d, 2H, Ph, J ¼ 8.7 Hz);
7.62e7.68 (m, 3H, Ph); 7.82 (s, 1H, Ph); 7.86 (d, 2H, Ph, J ¼ 7.2 Hz);
7.93 (d, 2H, Ph, J ¼ 7.2 Hz); 8.02 (d, 2H, Ph, J ¼ 7.2 Hz); 8.14 (d, 2H,
J ¼ 6.9 Hz); 1.16e1.24 (m, 12H, CH2); 1.37e1.47 (m, 2H, CH2);
3.23e3.44 (m, 2H, CH2); 4.64e4.65 (m, 2H, H-50); 4.77e4.81 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.94e6.01 (m, 2H, H-20, H-30); 6.26 (d, 1H, H-
10, J ¼ 4.5 Hz); 7.41e7.51 (m, 6H, Ph); 7.57 (d, 2H, Ph, J ¼ 8.7 Hz);
7.62e7.69 (m, 3H, Ph); 7.82e7.87 (m, 3H, Ph); 7.93 (d, 2H, Ph,
J ¼ 6.9 Hz); 8.02 (d, 2H, Ph, J ¼ 7.2 Hz); 8.14 (d, 2H, Ph, J ¼ 8.7 Hz);
11.64 (s, 1H, NH). MS m/z Calc. for C46H47N3O12: 833.90, found
832.39 [M ꢃ H]ꢃ.
Ph, J ¼ 8.7 Hz); 11.64 (s, 1H, NH). MS m/z Calc. for C45H45N3O12
:
819.87, found 818.27[M ꢃ H]ꢃ.
4.1.3.2. Isomer (6h). Yield 205.0 mg (16%), m.p. 60e62 ꢁC,
4.1.4.2. Isomer (6i). Yield 213.2 mg (16%), m.p. 54e55 ꢁC,
[
a]
25 þ 56.8 (c 0.059, CHCl3); 1H NMR (DMSO-d6):
d
0.78e0.83 (m,
[a
]
25 þ 56.4 (c 0.078, CHCl3); 1H NMR (DMSO-d6):
d 0.81 (t, 3H, CH3,
D
D
3H, CH3); 1.18e1.26 (m, 10H, CH2); 1.41e1.52 (m, 2H, CH2);
3.25e3.45 (m, 2H, CH2); 4.62e4.65 (m, 2H, H-50); 4.73e4.78 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.95e6.02 (m, 2H, H-20, H-30); 6.22 (d, 1H, H-
10, J ¼ 3.6 Hz); 7.42e7.52 (m, 6H, Ph); 7.56e7.68 (m, 5H, Ph); 7.80 (s,
1H, Ph); 7.88e7.91 (m, 4H, Ph); 8.01 (d, 2H, Ph, J ¼ 7.5 Hz); 8.14 (d,
J ¼ 6.9 Hz); 1.18e1.24 (m, 12H, CH2); 1.42e1.51 (m, 2H, CH2);
3.25e3.46 (m, 2H, CH2); 4.62e4.64 (m, 2H, H-50); 4.73e4.77 (m, 1H,
H-40); 5.34 (s, 1H, CH); 5.95e6.02 (m, 2H, H-20, H-30); 6.22 (d, 1H, H-
10, J ¼ 3.6 Hz); 7.42e7.52 (m, 6H, Ph); 7.58e7.68 (m, 5H, Ph); 7.80 (s,
1H, Ph); 7.87e7.91 (m, 4H, Ph); 8.01 (d, 2H, Ph, J ¼ 8.4 Hz); 8.14 (d,
2H, Ph, J ¼ 8.7 Hz); 11.64 (s, 1H, NH). MS m/z Calc. for C45H45N3O12
:
2H, Ph, J ¼ 9.0 Hz); 11.64 (s, 1H, NH). MS m/z Calc. for C46H47N3O12
:
819.87, found 818.29 [M ꢃ H]ꢃ.
833.90, found 832.40 [M ꢃ H]ꢃ.
4.1.4. 20,30,50-tri-O-benzoyl-
b
-D-ribofuranosyl-5-[nonyl-(4-nitro-
4.1.5. General procedure for preparation of
b-D-ribofuranosyl-5-
phenyl)-methyl]-pyrimidine-2,4-dione (4i), (5i), (6i)
[alkoxy-(4-nitro-phenyl)-methyl]-pyrimidine-2,4-diones (7,8)
5-[Nonyl-(4-nitro-phenyl)-methyl]-1H-pyrimidine-2,4-dione
Nucleosides 5fe5i and 6fe6i were dissolved in MeOH/NH3
solution (7 ml) and stirred at room temperature for 6 days. Then the
mixture was evaporated, co-evaporated with methanol and purified
by silica gel column chromatography using CHCl3/MeOH (9/0.5).
(3i) (600 mg, 1.5406 mmol) was heated in hexamethyldisilazane
(15 ml) at 140 ꢁC with (NH4)2SO4 (approximately 5 mg) for 8 h.
After that it was evaporated and residue was dissolved in anhy-
drous 1,2-dichloroethane (20 ml). To this solution,1-O-acetyl-2,3,5-
tri-O-benzoyl-
methylsilyl trifluoro-methanesulfonate (307
b
-
D
-ribofuranose (777.2 mg, 1.5406 mmol) and tri-
4.1.5.1. (þ)-
b-D-ribofuranosyl-5-[hexyl-(4-nitro-phenyl)-methyl]-
pyrimidine-2,4-dione (7f). Nucleoside 7f was prepared from 5f
ml, 1.6962 mmol) were
added. This mixture was stirred at room temperature for 2 days,
washed with water (20 ml) and ethyl acetate (60 ml), dried over
sodium sulphate, filtered and evaporated to dryness. Yield of crude
product (4i) (mixture of diastereoisomers) 721.3 mg (54%). Two
diastereoisomers (5i) and (6i) were isolated in this order from the
mixture by silica gel column chromatography using methanol in
chloroform (0e5%).
(171.8 mg, 0.2170 mmol) according to general procedure.
Yield 83.1 mg (80%), m.p. 72e74 ꢁC, [
a
]
25 þ 55.2 (c 0.077, CHCl3);
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 7.2 Hz); 1.23e1.35 (m, 6H,
CH2); 1.49e1.58 (m, 2H, CH2); 3.41e3.48 (m, 2H, CH2); 3.50e3.64
(m, 2H, H-50); 3.88e3.98 (m, 3H, H-20, H-30, H-40); 5.01 (t,1H, 50-OH,
J ¼ 4.8 Hz); 5.06 (d, 1H, 30-OH, J ¼ 4.2 Hz); 5.35e5.38 (m, 2H, 20-OH,
CH); 5.78 (d, 1H, H-10, J ¼ 5.4 Hz); 7.62 (d, 2H, Ph, J ¼ 9.0 Hz); 7.98
(s,1H, Ph); 8.18 (d, 2H, Ph, J ¼ 8.7 Hz); 11.44 (s,1H, NH). MS m/z Calc.
for C22H29N3O9: 479.49, found 478.21 [M ꢃ H]ꢃ.
Table 3
4.1.5.2. (ꢃ)-
b-D-ribofuranosyl-5-[hexyl-(4-nitro-phenyl)-methyl]-
pyrimidine-2,4-dione (8f). Nucleoside 8f was prepared from 6f
Antimicrobial activity of compounds 7hei and 8hei [MIC (
mM)].
Compounds Enterococcus Staphylococcus Staphylococcus Staphylococcus
(113.5 mg, 0.1433 mmol) according to general procedure.
faecalis
aureus
aureus (MRSA) haemolyticus
25
Yield 60.1 mg (88%), m.p. 68e69 ꢁC, [
a
]
ꢃ 39.0 (c 0.39,
CCM 4224
CCM 3953
D
CHCl3); 1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 6.9 Hz);
7h
7i
200
50
100
50
100
50
200
100
200
100
1.25e1.36 (m, 6H, CH2); 1.49e1.58 (m, 2H, CH2); 3.42e3.50
(m, 2H, CH2); 3.52e3.56 (m, 2H, H-50); 3.84e3.86 (m, 1H, H-40);
3.95e3.99 (m, 1H, H-30); 4.02e4.08 (m, 1H, H-20); 5.02 (t, 1H,
8h
8i
200
50
100
50
100
50

L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
3593
50-OH, J ¼ 4.5 Hz); 5.10 (d, 1H, 30-OH, J ¼ 5.1 Hz); 5.39 (s, 1H, CH);
5.42 (d, 1H, 20-OH, J ¼ 5.4 Hz); 5.81 (d, 1H, H-10, J ¼ 5.1 Hz); 7.61
(d, 2H, Ph, J ¼ 8.7 Hz); 7.94 (s, 1H, Ph); 8.19 (d, 2H, Ph, J ¼ 8.7 Hz);
11.44 (s, 1H, NH). MS m/z Calc. for C22H29N3O9: 479.49, found
478.25 [M ꢃ H]ꢃ.
(m, 2H, 20-OH, CH); 5.77 (d, 1H, H-10, J ¼ 4.8 Hz); 7.62 (d, 2H, Ph,
J ¼ 8.7 Hz); 7.99 (s, 1H, Ph); 8.19 (d, 2H, Ph, J ¼ 8.7 Hz); 11.44 (s, 1H,
NH). MS m/z Calc. for C25H35N3O9: 521.57, found 520.30 [M ꢃ H]ꢃ.
4.1.5.8. (ꢃ)-b-D-ribofuranosyl-5-[nonyl-(4-nitro-phenyl)-methyl]-
pyrimidine-2,4-dione (8i). Nucleoside 8i was prepared from 6i
4.1.5.3. (þ)-
b
-
D
-ribofuranosyl-5-[heptyl-(4-nitro-phenyl)-methyl]-
(186.0 mg, 0.2230 mmol) according to general procedure.
pyrimidine-2,4-dione (7g). Nucleoside 7g was prepared from 5g
Yield 84.4 mg (73%), m.p. 63e64 ꢁC, [
a
]
25 ꢃ 32.2 (c 0.38, CHCl3);
D
(207.6 mg, 0.2576 mmol) according to general procedure.
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 6.9 Hz); 1.22e1.31
25
Yield 95.7 mg (75%), m.p. 63e65 ꢁC, [
a]
þ 7.5 (c 0.30, CHCl3);
(m, 12H, CH2); 1.49e1.58 (m, 2H, CH2); 3.44e3.47 (m, 2H, CH2);
3.52e3.56 (m, 2H, H-50); 3.85e3.86 (m, 1H, H-40); 3.95e3.99
(m, 1H, H-30); 4.02e4.08 (m, 1H, H-20); 5.02 (t, 1H, 50-OH,
J ¼ 4.2 Hz); 5.10 (d, 1H, 30-OH, J ¼ 5.1 Hz); 5.38 (s, 1H, CH); 5.42
(d, 1H, 20-OH, J ¼ 5.7 Hz); 5.79 (d, 1H, H-10, J ¼ 5.1 Hz); 7.61 (d, 2H,
Ph, J ¼ 9.0 Hz); 7.94 (s, 1H, Ph); 8.19 (d, 2H, Ph, J ¼ 9.0 Hz); 11.43
(s, 1H, NH). MS m/z Calc. for C25H35N3O9: 521.57, found 520.26
[M ꢃ H]ꢃ.
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 7.2 Hz); 1.23e1.32
(m, 8H, CH2); 1.49e1.58 (m, 2H, CH2); 3.35e3.48 (m, 2H, CH2);
3.51e3.64 (m, 2H, H-50); 3.88e3.98 (m, 3H, H-20, H-30, H-40);
4.99e5.02 (m, 1H, 50-OH); 5.06 (bs, 1H, 30-OH); 5.35e5.38 (m, 2H,
20-OH, CH); 5.79 (d, 1H, H-10, J ¼ 4.8 Hz); 7.63 (d, 2H, Ph, J ¼ 8.7 Hz);
7.98 (s, 1H, Ph); 8.19 (d, 2H, Ph, J ¼ 8.7 Hz); 11.44 (s, 1H, NH). MS m/z
Calc. for C23H31N3O9: 493.52, found 492.31 [M ꢃ H]ꢃ.
4.1.5.4. (ꢃ)-
b
-
D
-ribofuranosyl-5-[heptyl-(4-nitro-phenyl)-methyl]-
4.2. Biological activity
pyrimidine-2,4-dione (8g). Nucleoside 8g was prepared from 6g
(232.4 mg, 0.2884 mmol) according to general procedure.
4.2.1. Cell lines
Yield 107.5 mg (76%), m.p. 71e72 ꢁC, [
a
]
25 ꢃ 40.0 (c 0.30, CHCl3);
CEM, A549, and K562 cell lines were purchased from the
American Tissue Culture Collection (ATTC). Paclitaxel/daunoru-
bicin resistant sublines of K562/CEM cells were prepared and
characterized in our laboratories. The human T-lymphoblastic
leukemia cell line, CEM, was used for routine screening of
compounds [19] 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, 2 mM glutamine,
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 6.9 Hz); 1.23e1.32
(m, 8H, CH2); 1.50e1.59 (m, 2H, CH2); 3.47e3.52 (m, 2H, CH2);
3.54e3.60 (m, 2H, H-50); 3.84e3.87 (m, 1H, H-40); 3.95e3.99
(m, 1H, H-30); 4.02e4.08 (m, 1H, H-20); 5.00 (t, 1H, 50-OH,
J ¼ 4.8 Hz); 5.09 (d, 1H, 30-OH, J ¼ 4.8 Hz); 5.39e5.41 (m, 2H, CH,
20-OH); 5.80 (d, 1H, H-10, J ¼ 5.1 Hz); 7.62 (d, 2H, Ph, J ¼ 8.7 Hz); 7.94
(s,1H, Ph); 8.19 (d, 2H, Ph, J ¼ 8.7 Hz); 11.44 (s,1H, NH). MS m/z Calc.
for C23H31N3O9: 493.52, found 492.22 [M ꢃ H]ꢃ.
100 U/mL penicillin, 100
and NaHCO3).
mg/mL streptomycin, 10% fetal calf serum,
4.1.5.5. (þ)-
b-D-ribofuranosyl-5-[oktyl-(4-nitro-phenyl)-methyl]-
pyrimidine-2,4-dione (7h). Nucleoside 7h was prepared from 5h
4.2.2. Cytotoxicity assay
(228.3 mg, 0.2785 mmol) according to general procedure.
Cell suspensions were prepared and diluted according to the
particular cell type and the expected target cell density
(2500e30,000 cells/well based on cell growth characteristics). Cells
Yield 96.0 mg (68%), m.p. 67e69 ꢁC, [
a
]
25 þ 9.5 (c 0.32, CHCl3);
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 7.2 Hz); 1.22e1.31
(m, 10H, CH2); 1.49e1.58 (m, 2H, CH2); 3.43e3.48 (m, 2H, CH2);
3.54e3.65 (m, 2H, H-50); 3.86e3.98 (m, 3H, H-20, H-30, H-40); 5.00
(t, 1H, 50-OH, J ¼ 5.1 Hz); 5.06 (m, 1H, 30-OH); 5.36 (d, 1H, 20-OH,
J ¼ 5.1 Hz); 5.38 (s, 1H, CH); 5.78 (d, 1H, H-10, J ¼ 4.8 Hz); 7.63 (d, 2H,
Ph, J ¼ 8.7 Hz); 7.98 (s, 1H, Ph); 8.19 (d, 2H, Ph, J ¼ 8.7 Hz); 11.43
(s, 1H, NH). MS m/z Calc. for C24H33N3O9: 507.55, found 506.23
[M ꢃ H]ꢃ.
were added by pipette (80
Inoculates were allowed a pre-incubation period of 24 h at 37 ꢁC
and 5% CO2 for stabilisation. Four-fold dilutions, in 20- L aliquots, of
mL) into 96-well microtiter plates.
m
the intended test concentration were added at time zero to the
microtiter plate wells.
All tested compounds were dissolved in 10% DMSO and
concentrations were examined in quadruplicate. 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 MTT. Aliquots (10 mL) of the MTT stock
solution were pipetted into each well and incubated for a further
1e4 h. After this incubation period the formazan produced was
4.1.5.6. (ꢃ)-
b-D-ribofuranosyl-5-[oktyl-(4-nitro-phenyl)-methyl]-
pyrimidine-2,4-dione (8h). Nucleoside 8h was prepared from 6h
(175.0 mg, 0.2134 mmol) according to general procedure.
Yield 76.9 mg (71%), m.p. 62e64 ꢁC, [
a
]
25 ꢃ 36.0 (c 0.38, CHCl3);
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 7.2 Hz); 1.23e1.32 (m,
dissolved by the addition of 100
m
L/well of 10% aq SDS (pH ¼ 5.5),
10H, CH2); 1.49e1.57 (m, 2H, CH2); 3.42e3.49 (m, 2H, CH2);
3.52e3.56 (m, 2H, H-50); 3.83e3.87 (m,1H, H-40); 3.95e3.99 (m,1H,
H-30); 4.02e4.08 (m, 1H, H-20); 5.02 (t, 1H, 50-OH, J ¼ 4.5 Hz); 5.10
(d, 1H, 30-OH, J ¼ 4.8 Hz); 5.38 (s, 1H, CH); 5.41 (d, 1H, 20-OH,
J ¼ 5.1 Hz); 5.80 (d, 1H, H-10, J ¼ 5.1 Hz); 7.61 (d, 2H, Ph, J ¼ 8.7 Hz);
7.95 (s, 1H, Ph); 8.18 (d, 2H, Ph, J ¼ 8.7 Hz); 11.44 (s, 1H, NH). MS m/z
Calc. for C24H33N3O9: 507.55, found 506.28 [M ꢃ H]ꢃ.
followed by a further incubation at 37 ꢁC overnight. The optical
density (OD) was measured at 540 nm with a Labsystem iEMS
Reader MF. Tumour cell inhibitory concentration (IC) was calcu-
lated using the following equation: IC ¼ (ODdrug-exposed well/mean
ODcontrol wells) ꢀ 100%. The IC50 value, the drug concentration lethal
to 50% of the tumour cells, was calculated from appropriate dose-
response curves.
4.1.5.7. (þ)-
b
-
D
-ribofuranosyl-5-[nonyl-(4-nitro-phenyl)-methyl]-
4.2.3. Apoptosis and cell cycle analysis by FACS
pyrimidine-2,4-dione (7i). Nucleoside 7i was prepared from 5i
CEM cells were treated with appropriate compound at
concentrations corresponding to 1ꢀ and 5ꢀ IC50 values (107/
(180.7 mg, 0.2167 mmol) according to general procedure.
Yield 89.0 mg (79%), m.p. 62e64 ꢁC, [
a
]
25 þ 7.7 (c 0.26, CHCl3);
537 mM, 29/144 mM and 26/130 mM) for 3, 6, 9 and 24 h. Following
D
1H NMR (DMSO-d6):
d
0.84 (t, 3H, CH3, J ¼ 6.9 Hz); 1.22e1.33 (m,
the incubation cells were pelleted, washed in PBS and fixed with
ice-cold 70% ethanol overnight at ꢃ20 ꢁC. Low molecular weight
apoptotic DNA was extracted in citrate buffer and RNA was cleaved
by RNAse (0.5 mg/ml). The DNA was stained by propidium iodide
12H, CH2); 1.48e1.57 (m, 2H, CH2); 3.42e3.50 (m, 2H, CH2);
3.54e3.64 (m, 2H, H-50); 3.88e3.98 (m, 3H, H-20, H-30, H-40); 5.02
(t, 1H, 50-OH, J ¼ 4.8 Hz); 5.07 (d, 1H, 30-OH, J ¼ 4.5 Hz); 5.37e5.38

3594
L. Brulíková et al. / European Journal of Medicinal Chemistry 45 (2010) 3588e3594
(0.1 mg/ml), and the cells were analyzed by flow cytometry using
a 488 nm single beam laser (FACSCalibur, Becton Dickinson).
0.05% NP-40 in PBS at room temperature with rotation, washed
once in cold 1% glycine in PBS. The cells were then washed with
PBS, incubated with propidium iodide (0.1 mg/ml) and RNAse A
(0.5 mg/ml) for 1 h at room temperature in the dark and finally
analyzed by flow cytometry using a 488 nm single beam laser
(FACSCalibur, Becton Dickinson).
4.2.4. BrdU incorporation and cell cycle analysis [2]
CEM cells were treated with appropriate compound at
concentrations corresponding to 1ꢀ and 5ꢀ IC50 values (107/
537
mM, 29/144 mM and 26/130 mM) for 3, 6, 9 and 24 h. The cultures
were fed a pulse of 10
m
M 5-bromo-20-deoxyuridine (BrdU) for
4.2.6. Antimicrobial activity
30 min at 37 ꢁC before harvesting. The cells were collected, washed
with PBS and fixed with ice-cold 70% ethanol overnight. Then cells
were washed with PBS, and resuspended in 2 M aq HCl for 30 min
at room temperature to denature their DNA. Following neutrali-
zation with 0.1 M Na2B4O7, pH 5, the cells were harvested by
centrifugation and washed with PBS containing 0.5% Tween-20 and
1% BSA. They were then stained with primary monoclonal anti-
Antimicrobial activity was determined by the using of the
minimum inhibitory concentration as the lowest concentration of
the test substance that inhibited the growth of the bacterial strain
after incubation for 24h in a thermostat at 37 ꢁC.
Acknowledgement
BrdU antibody (1 mg/ml) (Exbio, s.r.o., Prague, Czech Republic) for
30 min at room temperature, following the incubation cells were
washed with PBS and stained for 30 min at room temperature with
This study was supported by grants from the Ministry of
Schools, Youth and Education of the Czech Republic (MSM
6198959216, MSM 6198959223 and LC07107).
the secondary mouse IgG-FITC antibody (4 mg/ml) (Sigma Chemical
Co., Prague, Czech Republic). The cells were then washed with
PBS, incubated with propidium iodide (0.1 mg/ml) and RNAse
A (0.5 mg/ml) for 1 h at room temperature in the dark and finally
analyzed by flow cytometry using a 488 nm single beam laser
(FACSCalibur, Becton Dickinson).
References
[1] E. De Clercq, Med. Res. Rev. 28 (2008) 929.
[2] E. De Clercq, Future Virol. 3 (2008) 393.
[3] S.I. Khan, M.I. Dobrikov, B.R. Shaw, Nucleos. Nucleot. Nucl. 24 (2005) 1047.
[4] F.E. Onen, Y. Boum, C. Jacquement, M.V. Spanedda, N. Jaber, D. Scherman,
H. Myllykallio, J. Herscovici, Biorg. Med. Chem. Lett. 18 (2008) 3628.
[5] J. Kaminski, M. Pachulska, R. Stolarski, Z. Kazimierczuk, Tetrahedron 53 (1997)
2609.
[6] A.E.S. Abdel-Megied, M.S. Motawia, E.B. Pedersen, C.M. Nielsen, Heterocycles
34 (1992) 713.
[7] A.E.S. Abdel-Megied, E.B. Pedersen, C.M. Nielsen, Monatsh. Chem. 122 (1991)
59.
4.2.5. BrU incorporation, flow cytometric analysis of RNA synthesis
[3]
The cellular synthesis of RNA was analyzed by flow cytometry,
based on in vitro incorporation of the RNA precursor 50-bromour-
idine (BrU), followed by its immunocytochemical detection with
suitable cross reacting BrDU antibody. CEM cells were treated with
derivatives at concentrations corresponding to 1ꢀ and 5ꢀ IC50
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with primary monoclonal anti-BrdU antibody (1 mg/ml) (Exbio, s.r.
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following the incubation cells were washed with PBS and stained
for 30 min at room temperature with the secondary mouse
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IgG-FITC antibody (4
mg/ml) (Sigma Chemical Co., Prague, Czech
Republic). Then cells were fixed 15 min with 1% paraformaldehyde,