Synthesis and cytotoxic activity of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils in their racemic form

Article abstract of DOI:10.1016/j.bmcl.2007.09.022

The preparation of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils with alkyl chain lengths C₁-C₁₂ is described. The synthesis is based on the preparation of 5-[chloro-(4-nitro-phenyl)-methyl]-uracil and subsequent substitution of chlorine with appropriate alcohols. The resulting ethers were tested for their cytotoxic activity in vitro against five cancer cell lines. The compounds were less active in lung resistance protein expressing cell lines, suggesting the involvement of this multidrug resistant protein in control of the biological activity. Cytotoxic substances induced rapid inhibition of DNA and modulation of RNA synthesis followed by induction of apoptosis. The data indicate that the biological activity of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils depends on the alkyl chain length.

Full text of DOI:10.1016/j.bmcl.2007.09.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6647–6650
Synthesis and cytotoxic activity of various
5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils in their racemic form
a,
b,
b
a
´
´
´
´
´
Lucie Spacˇilova, Petr Dzˇubak, Marian Hajdu´ch, Sonˇa Krˇupkova,
a
´ˇa,*
Pavel Hradil and Jan Hlavac
aDepartment of Organic Chemistry, Faculty of Science, Palacky´ University, Trˇ. Svobody 8, 771 46 Olomouc, Czech Republic
bLaboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine,
Palacky´ University and Faculty Hospital in Olomouc, Pusˇkinova 6, 775 20 Olomouc, Czech Republic
Received 17 July 2007; revised 31 August 2007; accepted 5 September 2007
Available online 8 September 2007
Abstract—The preparation of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils with alkyl chain lengths C1–C12 is described. The
synthesis is based on the preparation of 5-[chloro-(4-nitro-phenyl)-methyl]-uracil and subsequent substitution of chlorine with
appropriate alcohols. The resulting ethers were tested for their cytotoxic activity in vitro against five cancer cell lines. The com-
pounds were less active in lung resistance protein expressing cell lines, suggesting the involvement of this multidrug resistant protein
in control of the biological activity. Cytotoxic substances induced rapid inhibition of DNA and modulation of RNA synthesis
followed by induction of apoptosis. The data indicate that the biological activity of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils
depends on the alkyl chain length.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Compounds derived from 5-alkyluracil are well known
for their biological activity. Some of them, for exam-
ple, have been described as suitable agents for treating
various diseases caused by excessive cell proliferation,
such as in the treatment of various cancers1 or treat-
ment of proliferative diseases mediated by second mes-
sengers.2 The most potent 5-alkyluracils inhibited the
proliferation of leukemia, lymphoma, and solid tu-
mor-derived cell lines at micromolar concentrations.3
Our research is focused on derivatives of 5-alkoxyura-
cils with anticancer activity. In this paper we describe
the synthesis and cytotoxic activity of 5-[alkoxy-(4-ni-
tro-phenyl)-methyl]-uracils having various alkyl chain
lengths.
In our hands the reaction does not lead to compound 6,
but rather to a mixture of 5-[chloro-(4-nitro-phenyl)-
methyl]-uracil 3 and 5,50-(4-nitrophenyl)-methyl-bis-
1H-pyrimidine-2,4-dione 4 instead. We successfully tried
to find reaction conditions for selective preparation of
each derivative (Scheme 2).
Derivative 4 has been prepared recently by nitration of
5,50-phenylmethylene-bis-uracil and patented in the
class of anti-ictogenic or anti-epileptogenic agents.5
The reaction of uracil with benzaldehyde is reported to
afford the appropriate derivative 2.4 If 4-nitrobenzalde-
hyde is used instead, 5-[hydroxy-(4-nitro-phenyl)-
methyl]-uracil 6 results as is described in the same pub-
lication (Scheme 1).
Keywords: Uracil; DNA; RNA; Apoptosis; Cytotoxic; Anticancer
activity.
*
Corresponding author. Tel./fax: +420 585634405; e-mail addresses:
hajduchm@gmail.com; hlavac@prfnw.upol.cz
These authors contributed equally to the work.
Scheme 1. Reaction of uracil with benzaldehyde and p-nitrobenzalde-
hyde according to Ref. 4.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.022

´ ˇ ´
L. Spacilova et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6647–6650
6648
Scheme 2. Reaction of uracil with p-nitrobenzaldehyde.
Figure 1. Cytotoxic activity of compounds 7a–7j and control agent
(carboplatin) as a function of chain length in LRP negative CEM and
K562-Tax leukemia cell lines.
The chlorine atom of derivative 3 is very reactive. When
treated with sodium azide the appropriate azide 5
results, with DMF/water the alcohol 6 is obtained and
with alcohols the desired ethers 7 are formed (Scheme 3).
The prepared compounds 4–7 were tested for their cyto-
toxic activity against five tumor cell lines under in vitro
conditions. Compounds 4, 5 were found to be quite
inactive with IC50 above 100 lM. However, the activity
of first prepared ethers differed. For this reason, we
decided to prepare a series of alkoxyderivatives 7 with
various alkyl chain lengths. Compounds with longer al-
kyl chains (heptyl 7g to dodecyl 7l) exhibited relatively
higher cytotoxic activity in CEM and K562-Tax leuke-
mia cells. In these lines, the only exception in the activity
trend is derivative 7f (Fig. 1).
In cell lines A549, CEM-DNR-B, and K-562, the cyto-
toxic activity also increases with increasing chain length
(7-9 carbons) and then decreases. Again, derivative 7f
exhibits exceptions in the activity trend (Fig. 2).
Figure 2. Cytotoxic activity of compounds 7a–7j and control agent
(carboplatin) related to chain length in LRP positive A549, CEM-
DNRB, and K562 cell lines.
Branching of alkyls reduces the activity, because
compounds 7m, n, and o are quite inactive.
For better understanding of the biological uniqueness of
compound 7f we have studied its activity in more detailed
analysis of the cell cycle, apoptosis, DNA and RNA syn-
thesis using CEM T-lymphoblastic leukemia cells.6,7 The
potency of compound 7f was compared to those of the
two most potent substances 7g and 7h, whose alkyl chain
lengths differ only by one or two carbon lengths, respec-
tively. Analyses were performed at equiactive concentra-
tions corresponding to 1· IC50 and 5· IC50.
Interestingly, the cytotoxic activity of the alkoxyderiva-
tives 7f–h was accompanied by rapid inhibition of DNA
synthesis at concentration 5· IC50 (Fig. 3).
However, at concentration 1· IC50 inhibition of DNA
synthesis is significant only for derivative 7f. Moreover,
there was an apparent increase of RNA synthesis in
compounds 7g and 7h but inhibition in 7f at 1· IC50,
while in 5· IC50 the total RNA synthesis was inhibited
in cells treated with any of the compounds. Based on
these data we hypothesize that the transient increase of
Scheme 3. Preparation of 5-[(alkoxy-(4-nitrophenyl)-methyl]-uracil 7
and other derivatives.

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L. Spacilova et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6647–6650
6649
Figure 3. Summary of RNA/DNA analysis for CEM cancer cell line
treated with compounds 7f–h. Data are expressed as a percentage of
6,7
positive cells in the total cellular population.
Figure 4. Summary of conventional cell cycle and apoptosis analysis
for CEM cancer cell line treated with compounds 7f–h. Data are
expressed as a percentage of cells with corresponding DNA content in
6,7
the total cellular population.
RNA synthesis at 1· IC50 in cells treated with 7g and 7h
is due to compensatory mechanisms, for example, trea-
ted cells are compensating insufficiency of macromole-
cules via increased RNA biosynthesis.
[alkoxy-(4-nitro-phenyl)-methyl]-uracils is controlled
by the multidrug resistance associated protein LRP.
Interestingly, the cytotoxic activity of substances 7g
and 7h in contrast to derivative 7f is active in LRP neg-
ative, but p-glycoprotein (PgP) positive K562-Tax cells.
This means that they were not influenced by expression
of multidrug resistance associated protein PgP, suggest-
ing activity of compounds in PgP overexpressing
tumors.8 However, the cytotoxic potency of all com-
pounds was reduced in CEM-DNR-bulk cells, which
are, in addition to LRP positivity, characterized by
overexpression of the multidrug resistance protein 1
(MRP) and decreased expression of topoisomerase IIa
gene.8 These data suggest that compounds 7f–h are
transported via LRP and may, directly or indirectly, tar-
get topoisomerase IIa. The involvement of MRP1
dependent transportation is not clear, since derivatives
were relatively potent in another MRP1 positive cell line
A549.8
All three compounds 7f–h induced apoptosis at 5· IC50
concentrations, but did not cause any significant cell cy-
cle alterations in treated CEM cells (Fig. 4). Interest-
ingly, at concentration 1· IC50 only 7f caused
significant apoptosis within 24 h. This finding rather
contrasts with the lower cytotoxic potency of compound
7f in 3-day cytotoxic MTT assay compared to structures
7g, 7h and suggests that chemical or metabolic stability
of 7f could explain the incoherent biological behavior of
the compound.
Cell lines A549, CEM-DNR-B, and K-562 were gener-
ally more resistant to this class of compounds. Indeed,
cell lines A549, CEM-DNR-B, and K-562, express con-
sistently significant concentrations of lung resistance
protein (LRP) but not other multidrug resistance associ-
ated proteins compared to CEM and K562-Tax cells.8
Thus, we hypothesize that the cytotoxic activity of 5-
In conclusion, the synthesized derivatives of 5-[(azido-
(4-nitrophenyl)-methyl]-uracil (5), 5-[hydroxy-(4-nitro-

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L. Spacilova et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6647–6650
6650
phenyl)-methyl]-1H-pyrimidine-2,4-dione (6), 5,50-(4-
nitrophenyl)-methyl-bis-1H-pyrimidine-2,4-dione (4) as
well as the 5-[(alkoxy-(4-nitrophenyl)-methyl]-uracil (7)
substituted with low alkyl chain did not exhibit any sig-
nificant cytotoxic activity against cancer lines. Activity
increases with chain lengths continuously in lines
CEM and K562-Tax. In ethers 7 with longer chains
(from nonyl to undecyl), the activity decreases in LRP
positive cell lines A549, K562, and CEM-DNR-B. Com-
pounds 7f–h, which were studied in the DNA/RNA syn-
thesis inhibition test, exhibited similar activity in
concentration 5· IC50, where the synthesis of both nu-
cleic acids was inhibited. Inhibition of de novo DNA
and RNA synthesis is the most probable reason for ra-
pid induction of apoptosis in treated cells.
Acknowledgment
This study was supported in part by the Ministry of
Education of the Czech Republic (MSM 6198959216
and LC07017).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.09.022.
References and notes
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Inc., 1997, Chapter 7; Unit 7.12.
At the concentration 1· IC50, only compound 7f effec-
tively inhibited both synthesis of DNA and RNA and
induced apoptosis. However, the structure was rela-
tively inactive in a 3-day cytotoxic assay. The excep-
tional behavior of the hexylderivative 7f may be
caused by chemical or metabolic stability or differen-
tial affinity to the multidrug resistance associated pro-
teins. Compounds 7g, 7h left the DNA synthesis
uninfluenced, but caused an increase in RNA synthe-
sis. This may be caused by promotion of compensa-
tory mechanisms. Therefore, the apoptosis at this
concentration was not apparent within the first 24 h
of treatment.
The bulky alcohols used to form ethers 7m–o did not en-
hance the activity to any noticeable extent.
The activity of individual enantiomers and nucleosides
derived from derivatives 7 is now under intensive exam-
ination and will be published separately.
8. Noskova, V.; Dzubak, P.; Kuzmina, G.; Ludkova, A.;
Stehlik, D.; Trojanec, R.; Janostakova, A.; Korinkova, G.;
Mihal, V.; Hajduch, M. Neoplasma 2002, 49, 418.