C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells

Article abstract of DOI:10.1016/j.ejphar.2011.09.024

The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.

Full text of DOI:10.1016/j.ejphar.2011.09.024

European Journal of Pharmacology 672 (2011) 30–37
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and Cellular Pharmacology
C(5) modified uracil derivatives showing antiproliferative and erythroid
differentiation inducing activities on human chronic myelogenous
leukemia K562 cells
Eleonora Brognara a, Ilaria Lampronti a, Giulia Breveglieri a, Alessandro Accetta b, Roberto Corradini b,
Alex Manicardi b, Monica Borgatti a, Alessandro Canella a, Chiara Multineddu a,
Rosangela Marchelli b, Roberto Gambari a,
⁎
a
BioPharmaNet, Department of Biochemistry and Molecular Biology, Molecular Biology Division, University of Ferrara, Via Luigi Borsari, 46-44100 Ferrara, Italy
Department of Organic and Industrial Chemistry, University of Parma, Parco Area delle Scienze, 17/A, 43100 Parma, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 August 2010
Received in revised form 30 August 2011
Accepted 7 September 2011
Available online 21 September 2011
The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds act-
ing by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthe-
sizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation
when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of
different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with
erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of
chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antipro-
liferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect with-
out activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown
by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not
show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge
this compound should be considered among the most efficient inducers of erythroid differentiation of
K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction
of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with
molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.
Keywords:
Erythroid differentiation
Tumor growth
Isoorotic acid derivative
Chronic myelogenous leukemia
Beta-thalassemia
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
terminal erythroid differentiation of this as well as other tumor cell
lines (Bianchi et al., 1999, 2000, 2001; Chiarabelli et al., 2003; Gambari
The pyrimidine system is a very important pharmacophor core of
naturally occurring and synthetic bioactive compounds, interacting
with the synthesis and function of nucleic acids, interfering with bio-
synthetic pathways and competing for the same binding sites of nat-
urally occurring pyrimidines (Lagoja, 2005). Modified pyrimidines
are currently used as drugs; for example fluorouracil has cytostatic ef-
fects and is currently used in cancer therapeutics, azidothymidine
(AZT) was the first applied drug for HIV treatment and bacimethrin
(4-amino-5-hydroxymethyl-2-methoxypyrimidine) is known as the
simplest pyrimidine antibiotics (Reddick et al., 2001).
and Fibach, 2007; Olivieri, 1996; Osti et al., 1997). K562 cells exhibit a
low proportion of hemoglobin-synthesizing cells under standard cell
growth conditions, but are able to undergo terminal erythroid differ-
entiation when treated with a variety of compounds, including short
fatty acids (Gambari and Fibach, 2007), 5-azacytidine (Gambari and
Fibach, 2007), mithramycin and chromomycin (Bianchi et al., 1999;
Fibach et al., 2003), cisplatin and cisplatin analogs (Bianchi et al.,
2000), tallimustine (Bianchi et al., 2001; Gambari and Fibach, 2007),
rapamycin (Fibach et al., 2006), everolimus (Zuccato et al., 2007),
psoralens (Lampronti et al., 2003a, b) and resveratrol (Bianchi et al.,
2009). Following erythroid induction, increase of expression of ε and
γ globin genes is observed, leading to accumulation of Hb Portland
(ζ2γ2) and Hb Gower 1 (ζ2ε2) (Gambari and Fibach, 2007).
The K562 cell line (Lozzio and Lozzio, 1975) has been proposed as a
very useful experimental system to identify antitumor compounds
(Lampronti et al., 2003a, b; Lampronti et al., 2006) acting by inducing
The design and production of antiproliferative molecules targeting
the K562 cell system might be of great interest for the development of
cocktails exhibiting applications in the treatment of chronic myelog-
enous leukemia. For instance smenospongine (Aoki et al., 2004a, b),
crambescidin 800 (Aoki et al., 2004a, b) and doxorubicin derivatives
⁎
Corresponding author at: Department of Biochemistry and Molecular Biology, Uni-
versity of Ferrara, Via Fossato di Mortara n.74, 44100 Ferrara, Italy. Tel.: +39 532
974443; fax: +39 532 974500.
E-mail address: gam@unife.it (R. Gambari).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.09.024

E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
31
(Szulawska et al., 2007) were reported as molecules of possible inter-
2.2. Cell culture conditions, proliferation assay and cell differentiation
est for inhibiting of chronic myelogenous leukemia cell growth, stim-
ulating terminal differentiation along the erythroid program. Some
molecules, such as Pivanex (an HDAC inhibitor) (Rabizadeh et al.,
2007) and a morpholine derivative of doxorubicin (Jakubowska et
al., 2008), are synergistic with the most common anti-chronic mye-
logenous leukemia agents, STI571 (Imatinib). In addition to synergis-
tic effects, molecules inducing differentiation might be of great
interest for treatment of Imatinib mesylate-resistant human chronic
myelogenous leukemia cell lines, as recently demonstrated for the
phytoalexin resveratrol (Puissant et al., 2008).
Recently we reported a novel class of C-5 linked N-1 alkylated ura-
cil dimers rationally designed to potentially interact with adenine in
biological systems. These pyrimidine derivatives have shown antipro-
liferative and erythroid differentiation activities toward human
chronic myelogenous leukemia K562 cells (Accetta et al., 2009). In
this paper we report a screening study on a set of different modified
C(5) uracil derivatives (Table 1) for the evaluation of their antiproli-
ferative effect in connection with erythroid differentiation pathways,
and for defining a new class of drugs candidates for the treatment of
chronic myelogenous leukemia.
The human K562 cells (Lozzio and Lozzio, 1975), obtained from the
American Type Culture Collection (Rockville, Md., USA), were main-
tained in a humidified atmosphere of 5% CO2 at 37 °C in suspension cul-
ture using RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented
with 10% fetal bovine serum (FBS; Analitical de Mori, Milan, Italy), 50
units/ml penicillin and 50 mg/ml streptomycin (Bianchi et al., 2000).
For the experiments, cell were seeded at an initial concentration of
3×104 cells/ml and cultured in the presence of increasing concentrations
of compounds. Non-treated cells were considered as control. Cell growth,
according to cell number/ml, was usually determined after 3, 4 and
5 days of culture, using a cell counter (Coulter Electronics, Hialeah, FL,
USA). These time points were selected because between days 3 and 5
untreated control K562 cells are on the log phase of cell growth (Bianchi
et al., 2000). All experiments were repeated at least 3 times. In order to
determine the effects on the erythroid differentiation, cells were seeded
at an initial concentration of 3×104 cells/ml and the proportion of benzi-
dine-positive cells was determined after 4–7 days of cell culture using a
solution containing 0.2% benzidine in 0.5 M glacial acetic acid (10%
H2O2) as previously described (Bianchi et al., 2001; Chiarabelli et al.,
2003). Benzidine positivity indicates the presence of intracellular
hemoglobin.
2. Materials and methods
2.3. Transfection of K562 cells with fluorescence protein genes under the
γ-globin and the β-globin gene promoters
2.1. Chemical synthesis of uracil compounds
Compounds 1, 2 and 4 were purchased from Sigma-Aldrich and
tested without further purification. Synthesis of compounds 5, 8, 9,
10 and 11 was previously reported in Accetta et al. (2009). All synthe-
ses started from uracil, thymine or 5-carboxyuracil (isoorotic acid) as
described schematically in the Results section. Full synthetic proce-
dures and characterization of compounds 3, 12, 13, 14, 15, 16, 17
and 18 are reported elsewhere (Accetta et al., 2010).
K562 cells were stably transfected with the pCCL.Promβ.HcRed1.
Promγ.EGFP, containing the green and red fluorescence protein (FP)
genes under the control of the γ-globin and β-globin gene promoters,
respectively (Guerrini et al., 2009; Lampronti et al., 2009). In this sys-
tem, increases in the green and red signals are consistent with γ-glo-
bin and β-globin gene promoter driven activity, respectively. To
determine the activity of chemical compounds in inducing the ex-
pression of γ-globin and β-globin genes, cells were seeded at 8000
cells/ml and treated with the appropriate concentration of the chem-
ical inducer. After 5 days of culture, cells were assayed for fluorescent
proteins expression. First of all they were analyzed under a fluores-
cence inverted microscope, using filters suitable for both green and
red FPs. The fluorescence intensity was then determined by fluores-
cence-activated cell sorting (FACS) analysis.
Table 1
Schematic description of compounds screened in the present study.
Compound R1
R2
R3
1
2
3
–H
–H
–H
–H
–CH3
–CH2N3
–H
–H
–H
2.4. FACS analysis
4
5
6
7
8
9
10
11
12
–CH2COOH CH3
–H
–H
–COPh
–COPh
–H
–H
–H
–H
–
For the determination of fluorescence intensity by FACScan (Becton
Dickinson, Franklin Lakes, NJ, USA), cells were harvested and washed.
Then 1×104 cells were analyzed by the CellQuest™ version 3.3 soft-
ware (Becton Dickinson, Franklin Lakes, NJ, USA), using the fl1 channel
to detect green fluorescence and fl3 channel to detect red fluorescence.
The results were expressed as median fold, i.e. the ratio between the
median fluorescence intensity values obtained in the presence and ab-
sence of treatment, respectively. A graphic presentation of data was fi-
nally obtained by histograms, showing the number of cells versus the
expressed fluorescence intensity (Salvatori et al., 2009).
–H
–COOCH3
–H
–H
–CH3
–H
–CH3
–CONHnC4H9
–CH3
–COOH
–CONHCH2Ph
–CH3
–nC8H17
–nC8H17
–nC8H17
–nC8H17
nC8H17
–H
13
–
–I
CH2COOtBu
–CH2COOH –I
14
15
–H
–H
–
–CH2N3
CH2COOtBu
2.5. mRNA measurements
16
17
18
–CH2COOEt –CH2N3
–CH2COOEt –CH2NH2
–CH2COOEt –CH2NHCO(2-
naphthyl)
–H
–H
–H
RNA was isolated from K562 cells and measured by reverse tran-
scription quantitative real-time polymerase chain reaction (qRT-PCR)
as described (Chiarabelli et al., 2003) using gene-specific double fluo-
rescence labeled probes in an ABI Prism 7700 Sequence Detection Sys-
tem version 1.7.3 (Applied Biosystems, Monza, Italy). The following
primer and probe sequences were used: α-globin forward primer, 5′-
CAC GCG CAC AAG CTT CG-3′; α-globin reverse primer, 5′-AGG GTC
ACC AGC AGG CAG T-3′; α-globin probe, 5′-FAM-TGG ACC CGG TCA
ACT TCA AGC TCC T-TAMRA-3′; γ-globin forward primer, 5′-TGG CAA
Name (alternative name): 1, uracil; 2, thymine (5-methyluracil); 3, 5-azidomethyluracil; 4,
(thymin-1-yl)acetic acid (1-(carboxymethyl)thymine); 5, methyl 5-uracilcarboxylate
(methyl isoorotate); 6, 3-benzoyluracil; 7, 3-benzoyl-1-methyluracil; 8 N-butyl-1-methyl-
5-uracilcarboxamide; 9, 1-octylthymine; 10, 1-octyl-5-uracilcarboxylic acid (1-octylisoorotic
acid); 11, N-benzyl-1-octyl-5-uracilcarboxamide; 12, 1,3-dioctylthymine; 13, t-butyl (5-
iodouracil-1-yl)acetate; 14, (5-iodouracil-1-yl)acetic acid; 15, t-butyl (5-azidomethyluracil-
1-yl)acetate; 16, ethyl (5-azidomehtyluracil-1-yl)acetate; 17, ethyl (5-aminomethyluracil-
1-yl)acetate; 18, ethyl [5-(N-(2-naphthylcarboxyl)aminomethyl)uracil-1-yl]acetate.

32
E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
Fig. 1. Synthetic pathways used for derivatives obtained from uracil. i) CH2O, Et3N, 60 °C, overnight; ii) HCl, 37 °C, 4 h, r.t.; iii) NaN3, DMF, r.t., 2 h; iv) BrCH2COOR; K2CO3, 0 °C to r.t.,
overnight; v) PPh3, THF, rt, overnight, then TFA; vi) a) BzCl acetonitrile/pyridine, r.t. 24 then b) K2CO3 , water/dioxane, r.t., 40 min; and c) NaHCO3 sat r.t. 1 h; vii) CH3I, DMF, r.t., 3 h.
GAA GGT GCT GAC TTC-3′; γ-globin reverse primer, 5′-TCA CTC AGC
TGG GCA AAG G-3′; γ-globin probe, 5′-FAM-TGG GAG ATG CCA TAA
AGC ACC TGG-TAMRA-3′; glycophorin A forward primer: 5′-CGG TAT
TCG CCG ACT GAT AAA-3′; glycophorin A reverse primer: 5′-AAA GGC
AGT CTG TGT CAG GT-3′; glycophorin A probe: 5′-FAM-AAA GCC CAT
CTG ATG TAA AAC CTC TTC CCC T-TAMRA-3′; transferrin receptor
forward primer: 5′-TCA GAGCGTCGGGATATCG-3′; transferrin receptor
reverse primer: 5′-TGA ACT GCC ACA CAG AAG AAC A-3′, transferrin
receptor probe: 5′-FAM-TGG CGG CTC GGG ACG GA-TAMRA-3′. The
kit for quantitative qRT-PCR for ζ-globin mRNA and ε-globin mRNA
was from Applied Biosystems (ζ-globin mRNA: Hs00923579_m1; ε-
globin mRNA: Hs00362216_m1). The fluorescent reporter and the
quencher were 6-carboxyfluorescein (FAM) and 6-carboxy-N,N,N′,N′-
tetramethylrhodamine (TAMRA), respectively. For qRT-PCR, the refer-
ence gene was 18S; this probe was fluorescent-labeled with VIC (Ap-
plied Biosystems, Monza, Italy) (Bianchi et al., 1999; Chiarabelli et al.,
2003).
Since these compounds are structurally analogs of modified nucleo-
sides, they could be considered as potential drugs per se.
The list of compounds tested is reported in Table 1. This set of
compounds was chosen for screening since: i) it contains compounds
alkylated at N1 and N3 positions or both; ii) it contains a series of C5
derivatives with different functional groups; iii) it has lipophilic
groups in both the N1, N3, or C5 region of uracil; iv) it contains deriv-
atives with intact uracil (and thymine) hydrogen bond pattern or
lacking of the N3 hydrogen.
The synthetic pathways used for different compounds are
reported in Figs. 1 and 2 (Accetta et al., 2009; Accetta et al., 2010).
Briefly, modification of uracil at both N1 and C5 positions was
obtained in several ways. For the more reactive uracil and isoorotic
acid, and in the presence of small electrophiles such as methyl iodide,
the regioselective alkylation at N1 can be obtained by temporary pro-
tection of the N3 with trimethylsilyl (Accetta et al., 2009) or benzoyl
(Frieden et al., 1998) groups. The latter method was used in the syn-
thesis of 7 from 6. Azidomethyl derivative 3 was obtained by reaction
of uracil with formaldehyde, thus introducing a 5-hydroxymethyl de-
rivative which could be further elaborated to azide by nucleophilic
substitution (via a chloromethyl intermediate). Alkylation of 3 with
α-bromoacetic acid derivatives led to compounds 15 and 16. The lat-
ter was converted to 17, bearing an amino group through Staudinger
reduction. The amide derivative 18 was then obtained by reaction of
the amino compounds with 2-naphthalenecarboxylic acid after acti-
vation of the latter with HBTU. Regioselective alkylation at N1 was
obtained also by exploiting the higher reactivity of the N1 position
in thymine and iodouracil toward sterically hindered electrophiles
3. Results
3.1. Synthetic scheme of the compounds tested
The C-5 position of pyrimidine nucleosides can be easily modified
with a variety of different residues. Substitution at this position is
often used for introducing substituents on DNA oligomers with re-
porter groups. In a general project, aimed at molecular engineering
of peptide nucleic acid (PNA) derivatives (A. Accetta, 2010), we pre-
pared a set of N(1) and/or N(3) alkyl-C(5) modified uracil derivatives.
Fig. 2. Synthetic pathways used for derivatives obtained from thymine and 5-iodouracil. i) For 9 Br-n-C8H17 , NaH as base, DMF, 80 °C, 4 h; for 13 BrCH2COOtBu and K2CO3 as base,
DMF; ii) 2,6-lutidine, K2S2O8, CuSO4, H2O/AcCN, 80 °C, 1,5 h; iii) NaClO2, NaH2PO4; t-BuOH/THF, room temp, 24 h).; iv) TFA, DCM, 0 °C, 1h.

E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
33
Table 2
3.2. Antiproliferative and erythroid differentiating activities of the uracil
monomers
a
Antiproliferative activity (IC50
cells) of uracil derived-compounds on erythroleukemia K562 cells line.
)
and induction of erythroid differentiationb (% B+
Compound
IC50
% B+ cells
Concentration
(μM)
We first determined for all the synthesized molecules the effects
on cell proliferation. To this aim, K562 cells were cultured in the pres-
ence of increasing concentrations of compounds and cell number/ml
was determined after 3, 4 and 5 days of treatment. The results are
shown in Table 2, where it appears evident that the most active com-
pounds with respect to antiproliferative effects (IC50 values lower
that 150 μM) are compounds 3, 9, 11, 12 and 18, compound 9 being
the most active molecule. However, among these molecules, only
compound 9 is a very effective inducer of erythroid differentiation
of K562 cells (Table 2). When analysis of the proportion of benzi-
dine-positive cell is performed, K562 cells cultured in the presence
of 50 μM compound 9 display over 90% hemoglobin-expressing (ben-
zidine-positive) cells. As far as the other compounds, only 8 and 18
are able to induce differentiation, but at very high concentrations
(400 μM and 200 μM, respectively). When the same compounds
were added at higher concentrations, no further increase in the pro-
portion of benzidine-positive cells was obtained (data not shown).
According with these results, compound 9 was analyzed in more
detail.
1
2
3
4
5
6
7
8
>400 μM
>400 μM
120.05 4.7 μM
>400 μM
>800 μM
350.7 15.9 μM
650.6 12.4 μM
488 68 μM
29.4 1.2 μM
268 15.5 μM
66.54 13 μM
29.7 1.4 μM
>400 μM
1.1 0.8
1.3 0.6
4.1 1.2
2.0 0.9
7.3 1.9
1.1 0.5
1.5 0.8
12 1.9
98 3.6
25 2.9
2.2 1.0
2.1 0.9
1.2 0.2
1.6 0.8
2.3 0.6
2.1 0.8
2.0 0.9
30 4.9
400
400
100
400
800
400
800
400
50
200
100
50
400
400
800
800
800
200
9
10
11
12
13
14
15
16
17
18
>400 μM
721.3 20.9 μM
764.2 25.6 μM
>800 μM
115 16 μM
a
Results are presented as average S.D. (three independent experiments performed).
Values are expressed as average S.D. (three independent experiments performed)
b
after 6 days of treatment at concentrations close to those needed to obtain 50% inhibition
of cell growth after 3 day culture period (and reported of the far right column).
3.3. Effects of compound 9 on proliferation and erythroid differentiation
of K562 cells
(Fig. 2), which allowed direct synthesis of compounds 9 (according to
Coutouli-Argyropoulou and Zachariadou, 2005) and 13. The N1, N3-
doubly alkylated compound 12 was obtained as a side product of thy-
mine alkylation. Oxidation of thymine methyl group led to the N1-
alkylated-5-carboxylic derivative 10 (Accetta et al., 2009), which
was then converted into the carboxyamide derivative 11 via HBTU ac-
tivation and reaction with benzylamine. Hydrolysis of 13 with TFA
provided derivative 14, bearing a polar substituent in the N1-position.
The analysis of the effects of compound 9 on the K562 cellular sys-
tem is shown in Fig. 3. In the experiment shown in Fig. 3A, K562 cells
were cultured for 6–7 days in the absence or in the presence of in-
creasing concentrations (10, 30, 50 μM) of compound 9. As it is clearly
evident, a concentration-dependent inhibition of cell growth was ob-
served. Fig. 3C reports the effect on the proliferation of K562 cells
after 3 days of treatment with compound 9 at different concentra-
tions. IC50 was obtained at about 30 μM. Fig. 3B shows the
A
B
1.5
100
75
50
25
0
1.0
0.5
0.0
0
1
2
3
4
5
6
0
1
2
3
4
5
6
7
days
days
C
D
1.2
1
100
75
50
25
0
0.8
0.6
0.4
0.2
0
(-)
10
30
50
(-)
10
30
50
concentration (µM)
concentration (µM)
Fig. 3. Effects of compound 9 on the proliferation (A) and erythroid differentiation of K562 cells (B). Cells were cultured for 6–7 days in the absence ( ) or in the presence of
increasing concentrations 10 μM ( ), 30 μM ( ), and 50 μM ( ) of compound 9. The effect on the proliferation of K562 after 3 days of treatment with compound 9 at different
concentrations (C) and the percentage of benzidine-positive cells after 6 days of treatment (D). The values in treated cultures represented in C) were compared with untreated
control cultures (taken as 100%). The data represents the mean S.D. of three independent experiments.

34
E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
Table 3
Schilsky, 1996). Interestingly, as far as the effects on cell proliferation,
Antiproliferative effects (IC50)
a and induction of erythroid differentiationb (% B+ cells)
similar IC50 values were obtained when compound 9 and 5-FU were
employed. On the other hand the effects of compound 9 and 5-FU
on erythroid differentiation were found to be very different, being
only compound 9 able to activate the full program of differentiation.
As expected, uracil, in agreement with the first set of results depicted
in Table 2, did not show any effects on K562 with respect to inhibition
of cell proliferation and induction of erythroid differentiation
(Table 3).
of uracil, compound 9 and 5-fluorouracil on K562 cell line.
Compound
IC50
% B+ cells
(30 μM)
Uracil
Compound 9
5-Fluorouracil
>500 μM
17.52 2.88 μM
17.73 2.23 μM
1.2 0.2
92.3 2.1
3.1 0.6
a
Results are presented as average S.D. (three independent experiments per-
formed) of concentration needed to obtain 50% inhibition of cell growth after 3 days
culture period.
3.4. Effects of compound 9 on the transcriptional activity of the γ-globin
and the β-globin gene promoters of K562 cells
b
Values are expressed as average S.D. (three independent experiments per-
formed) after 6 days of treatment.
To determine the effects of compound 9 on the transcription of the
globin genes, we used a clonal K562 cell population stably transfected
with a reporter construct, carrying the genes for the green (EGFP) and
red (RFP) fluorescent proteins (FP), under the control of the γ-globin
and the β-globin promoters, respectively (Guerrini et al., 2009; Lampronti
et al., 2009). To compare the relative effect of compound 9, the experi-
ment depicted in Fig. 4 was performed. Cells were treated with 30 μM
compound 9, isolated after 5 days and employed in FACS analysis; this
assay demonstrated that compound 9 enhanced EGFP by 2.10 0.63
fold, and the increase in RFP was 1.39 0.10 fold, indicating an effect of
compound 9 on the transcription directed by both the γ-globin and β-glo-
bin gene promoters. On the contrary, as recently published by our re-
percentages of differentiation induced by compound 9 at different
concentrations (10, 30, 50 μM). We like to underline that more than
60% benzidine positive cells are detectable after only 3 days of induc-
tion, demonstrating that the inducing effects of compound 9 was very
high, and it can be considered among the most powerful inducer of
K562 differentiation (see also Table 3). Despite the fact that there is
no linear relationship between proportion of differentiated cells and
cells with lower proliferation efficiency, the data summarized in
Fig. 3C and D demonstrate that 30 μM compound 9 is a concentration
sufficient to (a) inhibit cell growth and (b) induce differentiation of
the majority of treated K562 cells. Further experiments are required
to verify whether decrease of cell growth is a pre-requisite for differ-
entiation or is the cause of this feature. Supplementary Fig. 1 shows a
microscopic analysis of K562 cells cultured taken after 6 days of K562
cell cultured in the absence or in the presence of 30 μM compound 9.
In these assays, the blue-color of benzidine-stained cells is compatible
with high accumulation of hemoglobin (Bianchi et al., 2001).
search group, other erythroid differentiation inducers have
a
stimulatory selectivity only for the γ-globin gene promoter (Guerrini et
al., 2009 and data not shown).
3.5. Effects of compound 9 on biochemical parameters associated to the
activation of the K562 erythroid phenotype
In a second set of experiments, the biological activity of compound
9
was compared to that of 5-fluorouracil (5-FU), extensively
As we can see in Fig. 5, exposure for 6 days of K562 cells to 30 μM
compound 9 stimulates a sharp accumulation of α-globin mRNA
employed as anti-tumor agent in leukemias (Mini et al., 1990;
Fig. 4. Representative example of FACS analysis of a K562 cell clone containing the reporter construct pCCL.Promβ.HcRed1.Promγ.EGFP, untreated or treated with compound 9
30 μM for 5 days. (A,B) Histogram plots obtained from untreated (solid lines) or treated (dotted lines) cells, showing the relationship between number of cells and intensity of
expressed fluorescence, green (A) or red (B), respectively. (C) Dot plots obtained from untreated (−, upper panel) or treated (+, lower panel) cells, showing the cell population
distribution as a function of the two different fluorescences.

E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
35
A
B
C
60
16
12
20
16
12
8
45
30
8
α-globin
15
γ-globin
4
2
4
0
10
30
50
concentration (µM)
16
12
8
Fig. 6. Accumulation (fold increase in respect to control uninduced cells) of the α-like
ζ- and α-globin mRNAs, the β-like ε- and γ-globin mRNA, the glycophorin A and trans-
ferrin receptor mRNAs in K562 cells treated for 6 days with 30 μM compound 9. Results
represent the average S.D. of three independent experiments.
Further qRT-PCR data demonstrated that also the embryonic-type
ζ- and ε-globin mRNAs are induced by compound 9 (Fig. 6). In addi-
tion, the effects of compound 9 on other genes involved in the expres-
sion of erythroid program. To this aim the mRNA coding glycophorin
A (Ida et al., 2009; Kohmura et al., 2004; Watanabe et al., 1985) and
transferrin receptor (Gambari et al., 1986; Harigae et al., 2006) were
studied by qRT-PCR. The results obtained are shown in Fig. 6 and
demonstrate that both relative amounts of glycophorin A and trans-
ferrin receptor mRNAs increase following treatment with compound
9, confirms the induction of erythroid pathway by this compound.
α-globin
4
0
compd 9
30µM
compd 11
100µM
compd 10
100µM
20
16
12
8
3.6. The induction of erythroid differentiation of compound 9 is not
associated with the activation of the apoptotic pathway
Since several inducers of erythroid differentiation are also strong
inducers of apoptosis of K562 cells (for instance mithramycin and
psoralens) (Bianchi et al., 2001; Lampronti et al., 2003a, b) the exper-
iments described in Supplementary Figs. 3, 4 and 5 were performed.
Flow cytometry analysis (Supplementary Fig. 3) demonstrates no
major alteration of cell cycle parameters and no accumulation of
sub-G1 cells (a hallmark of apoptosis). Apoptosis was not evident
also when Annexin V–FITC staining was performed (Supplementary
Fig. 4) and using the DeadEnd TUNEL assay (Supplementary Fig. 5).
γ-globin
4
0
compd 9
30µM
compd 11
100µM
compd 10
100µM
4. Discussion
Fig. 5. Accumulation of α-globin mRNA (A, black columns) and γ-globin mRNA (A, gray
columns) in K562 cells treated for 6 days with 30 μM compound 9. qRT-PCR amplifica-
tions were performed on RNA from untreated or treated cells using primers amplifying
18S ribosomal RNA as reference gene. Results are presented as fold increase of α-globin
and γ-globin mRNAs with respect to untreated cells. (B,C) Effect of compounds 9, 11
and 10 on accumulation of α-globin mRNA (B) and γ-globin mRNA (C). The results
of untreated cells were taken as 1. Results represent the average S.D. of three inde-
pendent experiments.
The biological activity of the derivatives tested in this study can
lead to the following alternative effects: (a) a strong antiproliferative
effect linked to a high level of erythroid differentiation activity and
(b) an antiproliferative effect independent from the activation of
the erythroid program (Table 2). The highest antiproliferative effect
and erythroid induction ability were exhibited by compound 9, a thy-
mine derivative bearing a n-octyl chain on nitrogen N(1), whereas
thymine (compound 2) did not show any effect, suggesting the im-
portance of the linear alkyl chain in position N(1). The insertion of
the alkyl chain in both N(1) and N(3) positions of thymine (com-
pound 12) brought a loss of erythroid induction activity with reten-
tion of antiproliferative effect, suggesting the fundamental role of
hydrogen in position N(3) of thymine for the erythroid induction ac-
tivity. Appreciably, even if in some cases much lower, erythroid dif-
ferentiation activity was shown by derivatives 8, 10 and 18. Activity
of compound 10 can be rationalized considering its structural similar-
ities to compound 9 (methyl group substituted by COOH); com-
pounds 8 and 18 show the same pattern, when properly oriented
(black symbols) and γ-globin mRNA (gray symbols). The effects of
compound 9 on the transcription of α-globin mRNA and γ-globin
mRNA were analyzed by qRT-PCR amplifications and found to be
clearly associated with the ability of compound 9 to induce erythroid
differentiation of K562 cells. Compounds 10 and 11, which induce
erythroid differentiation to a much lower extent (Table 3), induce
only slight increase of accumulation of α-globin (Fig. 5B) and γ-glo-
bin (Fig. 5C) mRNAs. The qRT-PCR data concerning the effects of com-
pound 9 are fully in agreement with HPLC analyses, showing that this
molecule induces a sharp increase of embryonic and fetal hemoglo-
bins (Supplementary Fig. 2).

36
E. Brognara et al. / European Journal of Pharmacology 672 (2011) 30–37
Fig. 7. Structural similarities of the compounds most effective as erythroid inducers; compounds 18 and 8 are oriented as C-nucleoside analogs.
(Fig. 7); therefore, they can be considered as “C-nucleoside” equiva-
lent (Lu et al., 2009) of compound 9 with exchange of the role of N
(1) and C(5), which leads to N(3)-H in the same relative orientation
with respect to the lipophilic group as in thymine derivatives.
In conclusion we found a simple thymine derivative, N(1)-octyl-
thymine that exhibit strong antiproliferative activity, high ability to
induce terminal erythroid differentiation without activation of the ap-
optotic pathway. To our knowledge this compound should be consid-
ered among the most efficient inducers of erythroid differentiation of
K562 cells.
Italy (AVLT), and Telethon (grant GGP10214). We thank the EU Pro-
ject ITHANET (eInfrasctructure for Thalassemia Research Network)
for support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.ejphar.2011.09.024.
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