Synthesis and in vitro antiproliferative evaluation of novel N-alkylated 6-isobutyl- and propyl pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.04.079

A series of novel N-alkylated C-6-isobutyl- or -propyl pyrimidine derivatives were synthesized and their antiproliferative effect was evaluated on a panel of tumor cell lines including leukemia cell line K562 and normal diploid human fibroblasts. N-methox

Full text of DOI:10.1016/j.bmcl.2014.04.079

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro antiproliferative evaluation of novel
N-alkylated 6-isobutyl- and propyl pyrimidine derivatives
a,
b
c
b
a
⇑
´
´
´
´
Tatjana Gazivoda Kraljevic , Nataša Ilic , Višnja Stepanic , Lana Sappe , Jasna Petranovic ,
b,
⇑
a
´
´
´
´
Sandra Kraljevic Pavelic , Silvana Raic-Malic
a
´
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 19, HR-10000 Zagreb, Croatia
Department of Biotechnology, University of Rijeka, Trg bra´ce Mazurani´ca 10, HR-51000 Rijeka, Croatia
Division of Molecular Medicine, Ruder Boškovi´c Institute, Bijenicka 54, HR-10002 Zagreb, Croatia
b
c
ˇ
-
ˇ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-alkylated C-6-isobutyl- or -propyl pyrimidine derivatives were synthesized and their
antiproliferative effect was evaluated on a panel of tumor cell lines including leukemia cell line K562
and normal diploid human fibroblasts. N-methoxymethylated 5-methylpyrimidin-2,4-dione with di
(benzyloxy)isobutyl at C-6 (14b) showed the strongest effect on the cell growth at micromolar
concentrations. Mechanisms of action for the lipophilic compound 14b predicted in silico, pointed to
its anticancer and antimetastatic potential exerted through inhibition of DNA or RNA polymerases and
adhesion molecules. The latter mechanism has been supported in vitro for adherent tumor cell lines.
Ó 2014 Published by Elsevier Ltd.
Received 26 February 2014
Revised 16 April 2014
Accepted 21 April 2014
Available online xxxx
Keywords:
N-Alkylated pyrimidine derivatives
C-6-isobutyl pyrimidine
C-6-propyl pyrimidine
In silico analysis
Antiproliferative evaluation
Pyrimidines are integral DNA and RNA building blocks and thus
play an essential role in biological processes. As a consequence,
these compounds have considerable chemical and pharmacological
importance.1 Scaffolds containing the pyrimidine heterocycle exhi-
bit various biological effects including antiinflammatory, analgesic,
antipyretic, antiarrhythmic, antimicrobial, anticancer and antiviral
activities.2 As deoxynucleoside kinases are included in the activa-
tion of deoxynucleoside analogs with biological and therapeutic
properties, they are largely targeted for the treatment of viral
diseases and cancer.3 In particular, mitochondrial (mt) thymidine
kinase (TK-2) belongs to the family of mammalian deoxynucleo-
side kinases (dNKs) and its role is implicated in the phosphoryla-
tion of pyrimidine nucleosides required for the mitochondrial
DNA (mtDNA) whose synthesis is maintained during the whole cell
cycle.4 Besides the activation of nucleoside analogs with pharma-
cological properties, dNKs have a fundamental role in the salvage
pathway of deoxynucleotide synthesis. Extensive studies have
been carried out on the use of small side-chain 5-substituted uracil
analogs as enzyme inhibitors in cancer and viral chemotherapy by
phosphorylation with different efficacy using human and other
mammalian nucleoside kinases including thymidine kinases TK-1
and/or TK-2; viral kinases such as herpes simplex virus types 1
and 2 (HSV-1 TK and HSV-2 TK).5 Besides, C-6 fluoroalkylated
pyrimidines revealed pronounced cytostatic activities6 while thy-
mine with 6-(2,3-dihydroxypropyl) and 6-(1,3-dihydroxyisobutyl)
side-chain and its N-methylated structural analog have been devel-
oped as tracer molecules for monitoring HSV-1 TK expression by
positron emission tomography (PET).7 Moreover, it was found that
that N-1 and/or N-3-alkylated pyrimidine derivatives exert a wide
range of antiviral activity.8 Pronounced biological activities of C-5
and/or C-6 substituted pyrimidine derivatives substantiated by
previous reports in relation to their antiproliferative effects,
provide a good rationale for expanded study of the chemistry
and biological activities of this class.9 Furthermore, in chemother-
apeutical regimens pyrimidine nucleoside analogs are suitable for
combinational treatments with other cell cycle inhibitors targeting
different signaling pathways, mainly due to their DNA damaging
properties that induce the S-phase cell cycle arrest. Such approach
might enhance cytotoxicity of drugs and improve clinical response
of patients.3,10
In this study we report on the in vitro antiproliferative activity
and supposed mechanisms of biological action and toxicity of
pyrimidine derivatives substituted with acyclic side-chain at
position C-6 and alkylated with methoxymethyl (MOM) or trim-
ethylsilylethoxymethyl (SEM) group at N-1 and/or N-3 position of
pyrimidine ring. Synthesis of the key pyrimidine precursor 1a with
⇑
Corresponding authors. Tel.: +385 1 4597 215; fax: +385 1 4597 250 (T.G.K.);
tel.: +385 51 584 569; fax: +385 51 584 599 (S.K.P.).
E-mail addresses: tgazivod@fkit.hr (T. Gazivoda Kraljevic´), sandrakp@biotech.
uniri.hr (S. Kraljevic´ Pavelic´).
http://dx.doi.org/10.1016/j.bmcl.2014.04.079
0960-894X/Ó 2014 Published by Elsevier Ltd.
´
Please cite this article in press as: Gazivoda Kraljevic, T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.079

´
T. Gazivoda Kraljevic et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
2
6-(1,3-dibenzyloxy-2-hydroxyisobutyl) side-chain was performed
by treatment of 2,4-dimethoxy-6-methylpyrimidine with lithium
diispropylamide (LDA) in THF at ꢀ55 °C to afford the correspond-
ing lithiated precursor, which reacted in situ with 1,3-dibenzyl-
oxy-2-propanone to give 1a (Scheme 1). Deprotection of 2,4-
dimethoxy groups in 1a was accomplished using trimethylsilyl
iodide generated in situ from trimethylsilyl chloride and sodium
iodide to afford pyrimidin-2,4-dione 2a which was subsequently
submitted to N-alkylation reaction. Methoxymethylation reaction
of 2a with K2CO3 and methoxymethyl chloride in DMF afforded
N-1-MOM (3), N-3-MOM (4) and N,N-1,3-diMOM (5) 6-(1,3-diben-
zyloxy-2-hydroxyisobutyl)pyrimidine derivatives. N-1-SEM (6)
and N-3-SEM (7) regioisomers were obtained in reaction of
silylated pyrimidine derivative 2a with trimethylsilylethoxymeth-
yl chloride (SEMCl) in 1,1,1,3,3,3-hexamethyldisylazane (HMDS).
Debenzylation of N-1 regioisomers 3 and 6 with boron trichloride
gave N-1-MOM (8) and N-1-SEM (9) 6-(1,3-dihydroxy-2-hydroxyi-
sobutyl)pyrimidine derivatives, respectively. Synthesis of
N-MOM-5-methylpyrimidine derivatives 12b–14b was performed
according to the previously reported procedure starting from 6-
(1,3-dibenzyloxy-2-hydroxyisobutyl)-5-methylpyrimidine (1b).11
Reaction of C-5 unsubstituted pyrimidine derivative 1a with
methyl oxalyl chloride (MOC) gave oxalate, which was submitted
to Barton–McCombie deoxygenation using tributyltin hydride
(SnBu3H) and 2,20-azobis(isobutyronitrile) (AIBN) to give pyrimi-
dine derivative 10a containing di(benzyloxy)isobutyl side-chain
at C-6 (Scheme 2). Deprotection of dimethoxy groups in 10a was
accomplished with trimethylsilyl iodide to obtain pyrimidin-2,4-
dione 11a (Scheme 2). Methoxymethylation reaction of 11a with
MOMCl gave N-1-MOM (12a), N-3-MOM (13a) and N,N-1,3-
diMOM (14a) pyrimidine derivatives. Debenzylation of 12a and
14a was carried out using BCl3 to afford N-1-MOM (15) and
N,N-1,3-diMOM (16), respectively, with 6-(1,3-dihydroxyisobutyl)
side-chain. However, debenzylation of 14a accompanied by
intramolecular cyclization gave bicyclic compound 17 as minor
product (Scheme 2). Lithiation reaction of the 2,4-dimethoxy-6-
methylpyrimidine and its 5-methylated derivative, using LDA and
benzyloxyacetaldehyde in THF at ꢀ55 °C afforded 18a and 18b,
respectively, with C-6 3-benzyloxy-2-hydroxypropyl side-chain
(Scheme 3). Reaction of 18a and 18b with in situ formed trymeth-
ylsilyl iodide gave pyrimidine 2,4-dione derivatives 19a and 19b,
respectively, and 2,4-dimethoxypyrimidine 20b with free hydroxyl
functionality in C-6 side-chain. Reaction of silylated pyrimidine
derivatives 19a and 19b with SEMCl afforded N-1-SEM pyrimidine
derivatives 21a and 21b, respectively. Subsequently, debenzylation
of 21a and 21b with BCl3 gave target N-1-SEM pyrimidine deriva-
tives 22a and 22b, respectively, with free hydroxyl groups. Meth-
oxymethylation reaction of 19b with MOMCl gave N-1-MOM
(23b) and N,N-1,3-diMOM (24b) pyrimidine derivatives. Debenzy-
lation reaction of 23b afforded N-1-MOM (25b) pyrimidine with 6-
(1,3-dihydroxypropyl) side-chain.
Among 23 compounds tested for antiproliferative activity
in vitro, five compounds 2,4-dimethoxypyrimidine 1b and 10a
and pyrimidin-2,4-diones 12b, 13b and 14b with di(benzyloxy)iso-
butyl side-chain at C-6 (Scheme 2) showed antiproliferative effects
on five selected cancer cell lines and diploid fibroblast-like WI38 or
MCR cell lines (Table 1, Table S1 in Supplementary data). Among
C-5 unsubstituted pyrimidines, only non-N-alkylated 2,4-dimeth-
oxypyrimidine 10a exhibited cytostatic activity. Furthermore, in
the series of tested pyrimidin-2,4-dione derivatives, N-alkylated
N-1-MOM (12b), N-3-MOM (13b) and N,N-1,3-diMOM (14b)
5-methylpyrimidines displayed antiproliferative effect. The
strongest concentration-dependent effect on the growth of all
tested cells at micromolar concentrations (0.1–1 lM) was exerted
by N,N-1,3-diMOM pyrimidin-2,4-dione 14b. Therefore, antiprolif-
erative test for compound 14b was performed on the apoptosis
resistant non-adherent, Philadelphia chromosome-positive chronic
myeloid leukemia (Ph+ CML) cell line K562 in comparison with
commercial kinase inhibitor nilotinib (TasignaÒ). TasignaÒ is used
in standard treatment of Ph+ leukemia due to increased potency,
decreased toxicity and greater cellular and tissue penetration than
imatinib (GleevecÒ), with a similar target profile.14 Here presented
results showed higher antiproliferative effect of compound 14b on
K562 leukemia cells in comparison with TasignaÒ (Table 2) even
though TasignaÒ exhibited lower cytotoxicity on normal human
fibroblasts (BJ).
O
O
N
O
O
OBn
OBn
OBn
OH
HN
O
N
O
+
N
HN
(v)
+
OBn
OBn
OBn
OH
O
N
O
O
N
H
O
N
HO
HO
O
HO
HO
O
O
4
8
5
3
(iii)
OCH3
OCH3
O
N
OBn
OBn
N
HN
(ii)
(i)
H3CO
BnO
N
O
CH3
OBn
OBn
OBn
H3CO
N
O
N
H
HO
HO
1a
2a
(iv)
O
O
O
OH
Si
OBn
Si
OBn
HN
HN
O
N
(v)
+
OH
OBn
OBn
O
N
O
N
HO
HO
O
N
H
O
O
HO
Si
9
7
6
Scheme 1. Reagents and conditions: (i) LDA, THF, ꢀ55 °C, 20 h; (ii) TMSCl, NaI, rt, 19 h, Ar; (iii) K2CO3, DMF, MOMCl, rt; (iv) HMDS, SEMCl, reflux, 17 h; and (v) BCl3, CH2Cl2,
ꢀ55 °C, 4 h, Ar.
´
Please cite this article in press as: Gazivoda Kraljevic, T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.079

´
3
T. Gazivoda Kraljevic et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
OCH3
OCH3
N
R5
OBn
OBn
R5
OBn
OBn
(i)
N
N
H3CO
H3CO
N
10a,b
(ii)
HO
1a,b
R5
H
O
1a
R5
OBn
OBn
1b9b CH3
HN
O
N
H
11a,b
(iii)
O
N
O
N
O
R5
OBn
OBn
R5
OBn
OBn
R5
OBn
OBn
O
N
HN
O
N
+
+
O
O
O
N
H
O
O
13a,b
14a,b
12a,b
R5
(iv)
(iv)
O
H
10-14a
O
O
N
10-14b9b CH3
O
N
OH
OH
O
N
HN
+
O
N
OH
OH
O
N
O
O
O
O
OH
15
17
16
Scheme 2. Reagents and conditions: (i) MOC, DMAP, CH3CN, rt, 20 h; Bu3SnH, AIBN, toluene, reflux, 3 h; (ii) TMSCl, NaI, rt, 19 h, Ar; (iii) K2CO3, DMF, MOMCl, rt; and (iv) BCl3,
CH2Cl2, ꢀ55 °C, 4 h, Ar.
OCH3
R5
N
O
O
H3CO
N
R5
CH3
H
a
b
H
(i)
CH3
O
OCH3
OCH3
R5
R5
CH3
N
HN
OH
N
OH
+
(ii)
OH
OBn
OBn
OH
H3CO
N
O
N
H
H3CO
N
18a, b
20b
(iii)
(iv)
19a, b
O
O
O
R5
CH3
CH3
HN
OH
O
N
HN
OH
OH
+
OBn
OBn
O
OBn
N
O
N
O
N
O
O
O
Si
21a,b
(v)
23b
24b
(v)
O
O
R5
CH3
HN
HN
OH
OH
OH
OH
O
N
O
N
O
O
Si
22a,b
25b
Scheme 3. Reagents and conditions: (i) LDA, THF, ꢀ55 °C, 10 h; (ii) TMSCl, NaI, rt, 19 h, Ar; (iii) HMDS, SEMCl, reflux, 17 h; (iv) K2CO3, DMF, MOMCl, rt; and (v) BCl3, CH2Cl2,
ꢀ55 °C, 4 h, Ar.
´
Please cite this article in press as: Gazivoda Kraljevic, T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.079

´
T. Gazivoda Kraljevic et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
4
Table 1
The growth-inhibition effects of compounds 1b, 10a and 12b–14b on selected tumor
cell lines and normal human fibroblasts in vitro
a
Compd
IC50
(l
M)
MCF-7
SW620
MiaPaCa-2
HepG2
HeLa
WI38
MCR
1b12
10a
12b
13b
14b
4.2
0.3
72.6
21.6
0.4
3.5
6.1
NT
55.9
27.3
0.5
2.4
0.3
NT
NT
NT
3.7
3.5
29.7
17.0
0.3
3.8
NT
30.8
19.4
0.3
NT
18.5
NT
NT
NT
>100
72.1
25.6
0.5
a
IC50; 50% inhibitory concentration, or compound concentration required to
inhibit tumor cell proliferation by 50%. NT, non-tested. The cell growth rate was
evaluated by performing the MTT assay.13
Table 2
The inhibition effects of compound 14b and TasignaÒ on the growth of Ph+ CML cell
line K562 and normal human fibroblasts BJ in vitro
a
Compd
IC50
(
lM)
K562
BJ
TasignaÒ
14b
2.4
0.6
12.8
5.3
a
IC50; 50% inhibitory concentration, or compound concentration required to
inhibit tumor cell proliferation by 50%.
Observed antiproliferative effects might be partially explained
with relation to calculated n-octanol/water partition coefficients
logP. It has been revealed that the active compounds 1b, 10a,
12b–14b, all containing di(benzyloxymethyl) unit, are generally
more lipophilic than the inactive compounds (Table S2). The
greater lipophilic character may contribute to their considerable
cellular uptake and/or interactions with cellular target(s).15
According to PASS,16 a tool for estimation of potential pharma-
cological effects and biological targets, the synthesized molecules
were predicted to have antiviral activity (Pa P0.5 in Tables S3
and S4) what is in accordance with the observed antiviral activities
of some C-6 substituted thymine and uracil derivates.8 However,
different target molecules for pyrimidin-2,4-dione (Table S3) and
2,4-dimethoxypyrimidine derivatives (Table S4) were suggested
as the most probable by PASS. Although both classes have shown
antiproliferative effects (Table 1), their modes of action at molecu-
lar level may differ according to PASS.
Inhibitions of DNA and RNA polymerases as well as cell
adhesion molecules (Pa ꢁ0.3, Pi ꢁ0.1) were indicated for
pyrimidin-2,4-diones 12b, 13b and 14b (Table S3). On the contrary,
compounds 1b and 10a with 2,4-dimethoxypyrimidine scaffold
were predicted to have membrane permeability inhibitor capacity
(Pa >0.4; Pi ꢁ0.15) and to be Hsp 27 (b1) antagonist (Pa ꢁ0.5, Pi
ꢁ0.018) (Table S4). Since the most active compound 14b was indi-
cated as cell adhesion molecule inhibitor (Pa ꢁ0.4; Pi ꢁ0.05) and
antimetastatic agent (Pa ꢁ0.5, Pi ꢁ0.03) (Table S3), this compound
was subsequently tested by use of adhesion assay (Fig. 1).
Predicted inhibition of cell adhesion might explain the strong
antiproliferative effect observed on adherent tumor cell lines
(Table 1). It has already been shown that binding of tumor cells
to extracellular matrix proteins might promote tumor invasive-
ness, that is, binding of metastatic colon cancer cells to fibrinogen
or breast cancer cells to fibronectin.17 Adhesion analysis for adher-
ent tumor cells on the fibronectin matrix confirmed that adhesion
molecules are indeed, targeted by compound 14b (Fig. 1). It was
evident that adhesion of all tested cell lines was strongly affected
by treatment with compound 14b.
Figure 1. The results of adhesion assay. Tumor cell lines HeLa, MCF-7, SW620 and
normal human fibroblast BJ were treated with 14b at 5 M for (a) 2 h and (b) 24 h.
l
the lowest percentage of adhesion was observed for the metastatic
SW620 cell line (Fig. 1). Upon 24 h treatment with compound 14b
the strongest effect was detected on SW620 cells where almost no
cells were able to adhere to the matrix, while the weakest effect,
even though significant, was perceived on normal human fibro-
blasts (Fig. 1). These differences observed in the adhesion assay
of compound 14b were also visible in the cell cycle analysis
(Table 3).
In SW620 cells with mutated tumor-suppressor gene p53,18 an
increase of cells arrested in the S and G2/M phases has been
observed upon 24 and 48 h treatment with compound 14b instead
of standard chemotherapy-induced G1 arrest which occurs in p53
functional cells.19 Indeed, in normal human fibroblasts with wild
type (wt) p53 gene treated with compound 14b the arrest of cells
occurred in G1 and ended in subG1 arrest which is indicative of
apoptosis. A similar effect has been observed in MCF-7 cells bear-
ing a wt p5320 after 48 h treatment, where higher concentrations of
compound 14b induced an increase of cells in the G1 phase. Inter-
estingly, compound 14b showed no effect on the cell cycle of leu-
kemia cell line K562 even though it strongly inhibited their growth
(Table 4). Contrary, TasignaÒinduced a strong G1 arrest of K562
cells (at significance p < 0.05) that is in accordance with the litera-
ture data.20 Therefore, it may be assumed that compound 14b
exerts its antiproliferative effect on leukemia cells by some other
mechanism such as inhibition of DNA or RNA polymerases or
through a toxic effect on the cell membrane due to its lipophilic
properties.21
In conclusion, novel N-alkylated C-6-isobutyl or -propyl
pyrimidine derivatives were successfully synthesized. Lipophilic
2,4-dimethoxypyrimidine (1b and 10a) and N-1-MOM (12b),
N-3-MOM (13b) and N,N-1,3-diMOM (14b) pyrimidin-2,4-
dione derivatives all with di(benzyloxy)isobutyl side-chain at C-6
Prior to treatment with compound 14b, the adhesion rates of
tested cell lines were different and the highest percentages of
adhesion were observed for normal fibroblasts BJ and MCF-7, while
´
Please cite this article in press as: Gazivoda Kraljevic, T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.079

´
5
T. Gazivoda Kraljevic et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
Cell cycle analysis for compound 14b on adherent tumor cell lines
Cell line/treatment
Cell cycle phase (%)
subG1
G1
S
G2/M
subG1
G1
S
G2/M
subG1
G1
S
G2/M
SW620
WI38
MCF-7
Controla
0.1
Ma
Ma
Controlb
0.1
Mb
Mb
6.4
4.3
5.2
5.0
6.8
6.7
56.1
17.1
19.6⁄
16.1
21.8
25.6⁄
22
18.1
21.7⁄
20.1
14.4
15.5
15.8
36.7
21.9⁄
21⁄
27.9
38.5⁄
47.9⁄
41.7
11.8
8.9⁄
2.4⁄
8.6
9.1
50.2
55.5
23.5
25.5
13.7
9.7
8.1
7.1
8.7
10.3
9.5
3.8
l
51.5⁄
55.9
57.6
49.7⁄
53.1
61.7⁄
70.1⁄
53.9
6.9⁄
5.1⁄
8.7
1
l
67.1⁄
73.97
50.5⁄
78.9
14.8⁄
13.6
23.8⁄
11.5
8.1⁄
6.4
l
48.5⁄
45.5⁄
6.8
5.7
11.9⁄
4.9
1
l
3.4⁄
3.5⁄
a
24 h Treatment.
48 h Treatment. The results are shown as percentage of cells in a particular cell cycle phase (%). Statistically relevant results (p > 0.05) are denoted with an asterisk (⁄).
b
Table 4
2. (a) Amin, K. M.; Hanna, M. M.; Abo-Youssef, H. E.; George, R. F. Eur. J. Med.
Chem. 2009, 44, 4572; (b) Kumar, N.; Chauhan, A.; Drabu, S. Biomed.
Pharmacother. 2011, 65, 375; (c) Keri, R. S.; Hosamani, K. M.; Shingalapur, R.
V.; Hugar, M. H. Eur. J. Med. Chem. 2010, 45, 2597; (d) Bryzgalov, A. O.; Dolgikh,
M. P.; Sorokina, I. V.; Tolstikova, T. G.; Sedova, V. F.; Shkurko, O. P. Bioorg. Med.
Chem. Lett. 2006, 16, 1418; (e) Al-Abdullah, E. S.; Al-Obaid, A. R.; Al-Deeb, O. A.;
Habib, E. E.; El-Emam, A. A. Eur. J. Med. Chem. 2011, 46, 4642; (f) Kim, S.; Park, D.
H.; Kim, T. H.; Hwang, M.; Shim, J. FEBS J. 2009, 276, 4715.
3. Van Rompay, A. R.; Johansson, M.; Karlsson, A. Pharmacol. Ther. 2003, 100, 119.
4. Hernandez, A.-I.; Familiar, O.; Negri, A.; Rodriguez-Barrios, F.; Gago, F.;
Karlsson, A.; Camarasa, M.-J.; Balzarini, J.; Perez-Perez, M.-J. J. Med. Chem.
2006, 49, 7766.
Flow cytometric analysis of the cell cycle of K562 cells upon the treatments with
compound 14b
Compd 14b
Cell percentage (% standard deviation)
subG1
G1
S
G2/M
Controla
2.4 0.0
2.2 0.3
3.1 0.0
4.7 0.6
3.0 0.2
4.2 1.6
49.5 3.1
48.4 5.1
53.0 1.3
47.5 2.2
49.8 0.6
51.8 1.5
27.5 5.1
28.6 7.1
26.6 5.5
24.0 2.9
24.1 0.0
25.9 7.0
21.7 2.4
22.2 0.4
18.4 3.9
23.4 1.0
23.5 0.3
17.4 6.5
0.1
l
Ma
1
l
Ma
Controlb
0.1
Mb
Mb
l
1
l
5. (a) Kundu, N. G.; Mahanty, J. S.; Spears, C. P. Bioorg. Med. Chem. Lett. 1996, 6,
TasignaÒ
subG1
G1
S
G2/M
1497; (b) Kumar, R.; Nath, M.; Tyrrell, J. J. Med. Chem. 2002, 45, 2032.
´
6. (a) Prekupec, S.; Makuc, D.; Plavec, J.; Šuman, L.; Kralj, M.; Pavelic, K.; Balzarini,
Controla
1.8 0.3
3.7 0.2⁄
3.3 0.4⁄
3.0 0.7
48.4 0.1
85.0 1.1⁄
85.8 2.1⁄
51.2 2.4
70.0 1.7⁄
64.4 3.9⁄
28.9 0.9
7.0 1.1⁄
5.6 0.1⁄
21.7 2.0
16.5 0.3⁄
18.2 5.0⁄
21.8 0.5
4.4 0.1⁄
4.8 1.3⁄
26.1 0.1
5.1 1.2⁄
3.9 4.2⁄
J.; De Clercq, E.; Mintas, M.; Raic´-Malic´, S. J. Med. Chem. 2007, 50, 3037; (b)
Ma
Ma
´
Krištafor, S.; Gazivoda Kraljevic, T.; Makuc, D.; Plavec, J.; Šuman, L.; Kralj, M.;
0.1
0.5
l
l
Raic´-Malic´, S. Molecules 2009, 14, 4866; (c) Prekupec, S.; Makuc, D.; Plavec, J.;
Kraljevic´, S.; Kralj, M.; Pavelic´, K.; Andrei, G.; Snoeck, R.; Balzarini, J.; De Clercq,
Controlb
´
´
Mb
Mb
9.2 0.0⁄
15.0 3.1⁄
E.; Raic-Malic, S.; Mintas, M. Antiviral Chem. Chemother. 2005, 16, 327.
0.1
0.5
l
l
7. (a) Müller, U.; Martic´, M.; Gazivoda Kraljevic´, T.; Krištafor, S.; Ross, T. L.;
´
´
Ranadheera, C.; Müller, A.; Born, M.; Krämer, S. D.; Raic-Malic, S.; Ametamey, S.
a
M. Nucl. Med. Biol. 2012, 39, 235; (b) Krištafor, S.; Novakovic´, I.; Gazivoda
24 h Treatment.
b
ˇ
Kraljevic´, T.; Kraljevic´ Pavelic´, S.; Lucin, P.; Westermaier, Y.; Pernot, L.;
48 h Treatment. Statistically significant changes (p < 0.05) are denoted with an
´
´
Scapozza, L.; Ametamey, S. M.; Raic-Malic, S. Bioorg. Med. Chem. Lett. 2011,
21, 6161; (c) Raic´-Malic´, S.; Johayem, A.; Ametamey, S. M.; Batinac, S.; De
Clercq, E.; Folkers, G.; Scapozza, L. Nucleosides Nucleotides 2004, 23, 1707; (d)
Johayem, A.; Raic´-Malic´, S.; Lazzati, K.; Schubiger, P. A.; Scapozza, L.;
Ametamey, S. M. Chem. Biodivers. 2006, 3, 274.
asterisk (⁄).
showed antiproliferative activity on tested tumor cell lines. Among
these compounds, pyrimidin-2,4-dione derivate 14b exerted pro-
8. Loksha, Y. M.; Globisch, D.; Loddo, R.; Collu, G.; La Colla, P.; Pedersen, E. B.
ChemMedChem 2010, 5, 1847.
nounced inhibitory effect (IC50 ꢁ0.4
lines. Furthermore, this compound exhibited higher antiprolifera-
tive effect (IC50 = 0.6 M) on leukemia cell line K562 in comparison
with commercial kinase inhibitor TasignaÒ (IC50 = 2.4
M) that is
used in standard treatment of Ph+ leukemia. Pyrimidin-2,4-dione
derivatives 12b–14b have been recognized as antimetastatic
agents as they may target adhesion molecules, that was supported
in vitro for compound 14b on tumor cells.
lM) on all tested cancer cell
9. (a) Kundu, N. G.; Das, P.; Balzarini, J.; De Clercq, E. Bioorg. Med. Chem. 1997, 5,
2011; (b) Gazivoda Kraljevic´, T.; Krištafor, S.; Šuman, L.; Kralj, M.; Ametamey,
S. M.; Cetina, M.; Raic
l
´-Malic´, S. Bioorg. Med. Chem. 2010, 18, 2704.
l
10. Ibrahim, D. A.; El-Metwally, A. M. Eur. J. Med. Chem. 2010, 45, 1158.
´
´
11. Gazivoda Kraljevic, T.; Petrovic, M.; Krištafor, S.; Makuc, D.; Plavec, J.; Ross, T.
L.; Ametamey, S. M.; Raic´-Malic´, S. Molecules 2011, 16, 5113.
´
12. Krištafor, S.; Gazivoda Kraljevic, T.; Ametamey, S. M.; Cetina, M.; Ratkaj, I.;
ˇ
´
´
´
´
Tandara Hacek, R.; Kraljevic Pavelic, S.; Raic-Malic, S. Chem. Biodivers. 2011, 8,
1455.
´
´
13. Gazivoda, T.; Raic-Malic, S.; Krištafor, V.; Makuc, D.; Plavec, J.; Bratulic, S.;
Kraljevic´ Pavelic´, S.; Pavelic´, K.; Naesens, L.; Andrei, G.; Snoeck, R.; Balzarini, J.;
Mintas, M. Bioorg. Med. Chem. 2008, 16, 5624.
Acknowledgments
14. (a) Thomas, X. World J. Stem Cells 2012, 4, 44; (b) Sabha, N.; Au, K.; Agnihotri, S.;
Support of this study by the Ministry of Science, Education and
Sports of the Republic of Croatia (Projects #125-0982464-2925,
#335-0000000-3532 and #098-0982464-2511) is gratefully
acknowledged.
Singh, S.; Mangat, R.; Guha, A.; Zadeh, G. PloS One 2012, 7, e39412.
´
´
´
15. (a) Stepanic, V.; Koštrun, S.; Malnar, I.; Hlevnjak, M.; Butkovic, K.; Caleta, I.;
ˇ
Dukši, M.; Kragol, G.; Makaruha-Stegic´, O.; Mikac, L.; Ralic´, J.; Tatic´, I.; Tavcar,
´
B.; Valko, K.; Zulfikari, S.; Munic, V. J. Med. Chem. 2011, 54, 719; (b) Fantin, V. R.;
Berardi, M. J.; Scorrano, L.; Korsmeyer, S. J.; Leder, P. Cancer Cell 2002, 2,
29.
16. Filimonov, D. A.; Poroikov, V. V. In Chemoinformatics Approaches to Virtual
Screening; AlexandreVarnek, Alexander Tropsha, Eds.; RSC: Cambridge (UK),
2008; p 182.
17. (a) Alves, C. S.; Konstantopoulos, K. PloS One 2012, 7, e41472; (b) Bartsch, J. E.;
Staren, E. D.; Appert, H. E. J. Surg. Res. 2003, 110, 287.
18. Boyer, J.; McLean, E.; Aroori, S.; Wilson, P.; McCulla, A.; Carey, P.; Longley, D.;
Johnston, P. Clin. Cancer Res. 2004, 10, 2158.
19. (a) Amundson, S. A.; Myers, T. G.; Fornace, A. J., Jr. Oncogene 1998, 17, 3287; (b)
Bunz, F.; Hwang, P. M.; Torrance, C.; Waldman, T.; Zhang, Y.; Dillehay, L.;
Williams, J.; Lengauer, C.; Kinzler, K. W.; Vogelstein, B. J. Clin. Invest. 1999, 104,
263.
20. Lu, X.; Errington, J.; Curtin, N. J.; Lunec, J.; Newell, D. R. Clin. Cancer Res. 2001, 7,
2114.
Supplementary data
Supplementary data (procedures for syntheses of compounds
and their characterization data, methods for in silico analysis, anti-
tumor activity assay and cell cycle analysis) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.04.079.
References and notes
21. Wang, C.; Zhou, Y.; Li, S.; Li, H.; Tian, L.; Wang, H.; Shang, D. Life Sci. 2013, 92,
1004.
1. (a) Boncel, S.; Gondela, A.; Walczak, K. Curr. Org. Synth. 2008, 5, 365; (b) Amr, A.
E.; Ashraf, M. M.; Salwa, F. M.; Nagla, A. A.; Hammam, A. G. Bioorg. Med. Chem.
2006, 14, 5481.
´
Please cite this article in press as: Gazivoda Kraljevic, T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.079