New uracil dimers showing erythroid differentiation inducing activities

Article abstract of DOI:10.1021/jm800982q

The synthesis of C5 linked uracil dimers was carried out according to a model developed in order to bind adenine in DNA. N1-Alkylated uracil derivatives were synthesized from isoorotic acid (uracil-5-carboxylic acid) or thymine. The carboxylic acid deriva

Full text of DOI:10.1021/jm800982q

J. Med. Chem. 2009, 52, 87–94
87
New Uracil Dimers Showing Erythroid Differentiation Inducing Activities
Alessandro Accetta,† Roberto Corradini,*,† Stefano Sforza,† Tullia Tedeschi,† Eleonora Brognara,‡ Monica Borgatti,‡
Roberto Gambari,*,‡ and Rosangela Marchelli†
Dipartimento di Chimica Organica ed Industriale, UniVersita` degli Studi di Parma, Viale G.P. Usberti, 17/A, 43100 Parma, Italy, and
BioPhamaNet, Dipartimento di Biochimica e Biologia Molecolare, Sezione di Biologia Molecolare, UniVersita` di Ferrara, Via Luigi Borsari,
46- 44100 Ferrara, Italy
ReceiVed August 2, 2008
The synthesis of C5 linked uracil dimers was carried out according to a model developed in order to bind
adenine in DNA. N1-Alkylated uracil derivatives were synthesized from isoorotic acid (uracil-5-carboxylic
acid) or thymine. The carboxylic acid derivatives were condensed with diamines in order to produce dimeric
compounds or with monoamines in order to obtain reference monomeric compounds. Some of the derivatives,
in particular the uracil dimers, showed antiproliferative and erythroid differentiation induction properties
towards human chronic myelogenous leukemia K562 cells, thus indicating that these compounds could
represent a new class of drugs useful for the development of antitumor therapy based on the ability to
induce terminal differentiation.
Introduction
in the therapy of several hematological diseases, including
ꢀ-thalassemia and sickle cell anemia.14-20 K562 cells exhibit a
low proportion of hemoglobin-synthesizing cells under standard
cell growth conditions but are able to undergo erythroid
differentiation when treated with a variety of compounds,
including short fatty acids,19 5-azacytidine,19 mithramycin and
chromomycin,18,21cisplatinandcisplatinanalogues,17tallimustine,16,19
rapamycin,22 everolimus,23 psoralens,24 and resveratrol.25 Fol-
lowing erythroid induction, a sharp increase of expression of
human ε and γ globin genes is observed in K562 cells, leading
to a cytoplasmic accumulation of Hb Portland (ꢁ2γ2) and Hb
Gower 1 (ꢁ2ε2).19 Several antitumor drugs were demonstrated
to induce erythroid differentiation of K562 cells. Some of us
have recently demonstrated that DNA binding drugs (DBDsa)
exhibiting antitumor activity are powerful inducers of dif-
ferentiation of K562 cells, suggesting that the expression of
crucial genes involved in terminal erythroid differentiation
of these cells is influenced by DBDs.17,18,21 Several DBDs, such
as tallimustine, mithramycin, cisplatin, and angelicin, increase
fetal hemoglobin (HbF) production in erythroid precursor cells
from normal human subjects.19 Thus, this experimental cell
system appears to be suitable for the screening of molecules
able to inhibit cell growth by acting on the activation of terminal
differentiation pathways.
Pyrimidine derivatives are very important constituents of
naturally occurring bioactive compounds, including natural
nucleobases and their analogues.1 A series of pyrimidine
derivatives are currently used as drugs; for example, fluorouracil
has cytostatic effects and is currently used in cancer therapeutics,
and azidothymidine (AZT) was the first applied drug for HIV
treatment.
The potential of pyrimidine compounds is linked to the
possibility of being used as antagonists in the biosynthetic
pathways of pyrimidine nucleobases or in other important
processes, by competing for the same binding sites of naturally
occurring compounds. For example, oxime libraries based on
dimeric uracil derivatives have been proposed for the develop-
ment of uracil DNA glycosylase (UNG) inhibitors.2 5-Substi-
tuted uracil derivatives have been described as cytostatic and
antiviral compounds.3
Another possible use of pyrimidine analogues has recently
been explored in connection with the synthesis of tailor-made
modified nucleobases to be included in DNA analogues, such
as modified oligonucleotides4,5 or peptide nucleic acids (PNAs).6,7
For example, extended aromatic analogues of cytosine (named
“G-clamp”) bearing extra hydrogen bonding sites have been
shown to be able to strongly bind guanine by simultaneous
formation of Watson-Crick and Hoogsteen base pairing.8
The ability to bind to specific sites of DNA is also a
characteristic of many bioactive molecules able to act as
antibiotics antiproliferative or differentiating drugs. In this
respect, several in vitro experimental systems are available for
screening purposes. Among them, the K562 cell line, isolated
and characterized by Lozzio and Lozzio from a patient with
chronic myelogenous leukemia in blast crisis,9-11 has been
proposed as a very useful experimental system to identify (a)
antitumor compounds12,13 and (b) inducers of erythroid dif-
ferentiation and γ-globin gene expression of possible interest
In a general project aimed at the synthesis of oligonucleotide
analogues, in particular PNA, with modifications able to improve
their binding activity,26-28 we have designed uracil dimers
connected with a spacer through the 5-positions (Figure 1). The
design was performed by considering as a model the very stable
TAT triplet found in PNA/DNA/PNA triplex crystal structure.29
Since these modified nucleobases could also be considered as
potential drugs per se, they were subjected to a screening process
for the ability to induce erythroid differentiation and to exhibit
cytotoxicity, with positive results for some of the tested
compounds. In the present work we describe the synthesis of
these uracil dimers and the results obtained in the evaluation
of their differentiating properties, which allowed us to define a
* To whom correspondence should be addressed. For R.C.: phone, +39-
0521-905406; fax, +39-052-905472; e-mail, roberto.corradini@unipr.it. For
R.G.: phone, +39-0532974443; fax, +39-0532-974500; e-mail,
gam@unife.it.
a Abbreviations: AcCN, acetonitrile; AM1, Austin model 1; DBDs, DNA
binding drugs; DCM, dichloromethane; HbF, fetal hemoglobin; DIEA, N,N-
diisopropylethylamine; HMDS, hexamethyldisilazane; TMS-Cl, trimethyl-
silyl chloride; TFA, trifluoroacetic acid; Tr, trityl; UFF, universal force field.
†
Universita` degli Studi di Parma.
Universita` di Ferrara.
‡
10.1021/jm800982q CCC: $40.75
2009 American Chemical Society
Published on Web 12/15/2008

88 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Accetta et al.
Figure 1. Design of uracil dimers: (a) general scheme of uracil derivatives described in the present study, with numbering given according to
uracil nomenclature; (b) tautomeric forms of uracil-5-carboxamide moiety considered for calculations; (c) energy-minimized structures calculated
using the AM1 method and corresponding energies in kJ mol-1
.
new class of drug candidates, together with a rationale of the
possible connection between structure and activity based on their
design.
prone to exhibit the same recognition pattern of uracil as
originally designed.
Synthesis. In our retrosynthetic design, we used as starting
material either isoorotic acid (uracil-5-carboxylic acid) or
thymine. As a reference compound, isoorotic acid methyl ester
(1) was synthesized by reaction with SOCl2 in methanol. Several
derivatives were obtained with the purpose of varying the group
at N1, the type (monomeric or dimeric) of the amine residue,
and the size and rigidity of the spacer, in order to produce
chemical diversity.
Results and Discussion
Design. The general design of the uracil dimers is reported
in Figure 1a. Alkylation of uracil N1 is necessary either to
introduce lipophilic groups or for linking this compounds to
the backbone of nucleosides, nucleotides, or nucleotide ana-
logues. In all cases the N3 hydrogen was preserved in order to
maintain the same hydrogen-bond pairing scheme presented by
uracil. In the first part of this project, we designed a series of
dimers linked through amide bonds, since molecular modeling
(vide infra) indicated that the hydrogen bonding moieties could
be preserved using this kind of modification. Since the
compounds were designed in order to mimic and reinforce the
binding ability of uracil, we checked if the modification
introduced was able to induce severe changes in the uracil
potential binding sites, since electronegative substituents can
shift the tautomeric lactamic-lactimic equilibrium.30 The most
dramatic change in the structure was the introduction of a
carboxamide moiety in the 5-position of uracil; thus, we tested
the ability of these derivatives to be in the same tautomeric
forms, and hence with the same recognition pattern by hydrogen
bonds, by means of semiempirical calculation performed on the
model compound Mod-1 (Figure 1b).31 The structure of Mod-1
was first minimized by molecular mechanics using universal
force field (UFF) and then subjected to energy minimization
by a semiempirical method using the Austin Model 1 (AM1).
The same procedure was applied to the tautomer Mod-1(ii).
The most stable structures obtained in both cases are reported
in Figure 1c together with the corresponding enthalpies of
formation. The most stable structure Mod-1(i) has the amide
hydrogen pointing toward the C4 carbonyl oxygen, thus giving
rise to an intramolecular hydrogen bond. Both the structures
Mod-1(ii) and an alternative conformation Mod-1(iii) show
higher energies. Therefore, the 5-carboxamide compounds are
Direct alkylation of isoorotic acid with reactive substrates,
such as methyl or allyl halides, led to dialkylated products at
both nitrogen atoms (results not shown); therefore, regioselective
monoalkylation of uracil was performed using temporary
protection of the carboxylic and carbonyl oxygens with trim-
ethylsilyl groups, through reaction with hexamethyldisilazane
(HMDS) in a 3:1 excess and in the presence of trimethylchlo-
rosilane (TMS-Cl) (Figure 2a). This strategy not only allows
for monoalkylation, since the positively charged pyrimidinium
intermediate prevents further attack, but also directs the alky-
lation towards the less hindered N1 position. Confirmation of
the positions of the substituent in 2 and 3 was obtained by NOE
effects between the alkyl group and the CH(6) of uracil as
measured by 1D and 2D NOESY spectra.
Reaction of thymine with more hindered long chain primary
haloalkanes led to selective monoalkylation at N1 (Figure 2b),
thus affording the subtrate 4 suitable for the synthesis of uracil
derivatives with a C8 alkyl chain at N1. Oxidation of the methyl
group of 4 with K2S2O8 in the presence of copper(II) leads to
the uracil-5-carboxaldehyde, which was then oxidized by
reaction with sodium chlorite to the corresponding carboxylic
acid 5 bearing a C8 alkyl chain at the N1 position.
According to our model, the 2,7-di(aminomethyl)naphthalene
8 was a good candidate as a linking group for allowing
cooperative binding to adenine. Therefore, this compound was
synthesized using substitution of the corresponding bromide (6)

Uracil Dimers for Erythroid Differentiation
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 89
after 3, 4, and 5 days. These time points were selected because
between days 3 and 5 untreated control K562 cells are on the
log phase of cell growth. A representative experiment is shown
in Figure 5, in which the effects on cell growth (panels A and
C) and erythroid differentiation (panels B and D) of compounds
9 and 14 are compared. Compound 9 displays very low growth
inhibiting properties, while the IC50 for compound 14 was about
500 µM. Erythroid differentiation of the compounds under
investigation was studied by determining the proportion of
benzidine-positive (hemoglobin containing) cells. As clearly
evident in Figure 5 (panels B and D), compound 14 was found
to stimulate erythroid differentiation while compound 9 was not
active (Figure 5, panels A and B). Table 1 indicates the
antiproliferative effects (IC50 values) and the erythroid induction
ability (% of benzidine-positive cells) of all the tested com-
pounds. The best erythroid induction ability was displayed by
compound 14. The data shown in Table 1 were obtained using
concentrations of compounds approaching those giving 50% of
inhibition of cell growth (these concentrations were chosen to
better compare the potential erythroid inducing activity in
experimental conditions, leading for most of the compounds
tested, to similar effects on cell proliferation rate). In addition,
it should be noted that compound 14 was able to induce
erythroid differentiation of K562 cells after 6 days of cell culture
even if added at concentrations lower than that shown in Table
1 (an average of 52 ( 4.5% of benzidine-positive cells was
obtained in four independent experiments with 400 µM com-
pound 14). Under these experimental conditions no inhibition
of cell growth was detectable. In Table 2, the effect of compound
14 is compared with those of other erythroid inducers of K562
cells: the very high induction level suggests that 14 is indeed a
very active compound with activity comparable to those of other
previously reported compounds for this cell line.
The antiproliferative effect of compound 14 was obtained
using other tumor cell lines as cellular targets, including the
rhabdomyosarcoma RD33 and the two breast carcinoma MCF-
734 and MDA-MB-23135 cell lines (Table 3). Low antiprolif-
erative activity was found when the human cystic fibrosis
bronchial IB3-1 cell line36,37 was used (Table 3). As far as the
effects on K562 cell growth displayed by all the compounds
tested (Table 1), some compounds (for instance, compound 1
and 9) were found to be ineffective in inhibiting cell growth
under the experimental conditions employed; all the other
compounds were found to be moderately efficient in inhibiting
in vitro cell growth of treated K562 cells. As shown for
compound 14 (Table 3), the antiproliferative activity of the
compounds here studied is not restricted to K562 cells (data
not shown). Our results suggest that among the compounds
studied some are efficient at inducing erythroid differentiation
without exhibiting strong antiproliferative activity (i.e., com-
pound 14); conversely, some other compounds (i.e., compound
12) strongly inhibit cell growth of K562 cells without inducing
differentiation. When the experiments were conducted at
concentration higher than that reported in Table 1, compound
12 was confirmed to be inactive in inducing differentiation
(analysis was conducted at 200, 400, and 600 µM without any
effects in stimulating the increase of the proportion of benzidine
positive K562 cells). Further experiments employing proteomic
and transcriptomic analyses are necessary in order to understand
the interplay between effects of this class of molecules on cell
growth and erythroid differentiation.
Figure 2. Synthesis of N1-alkylated isoorotic acid derivatives (a) via
regioselective alkylation of Isoorotic acid ((i) HMDS,TMS-Cl, reflux
4 h; (ii) CH3I or ethyl bromoacetate in excess, reflux,18 h; (iii) H2O/
CH3COOH, room temp, 20 min.) and (b) via oxidation of thymine
derivatives ((iv) [Br-n-C8H17, NaH, DMF, 80 °C,4 h]; (v) 2,6-lutidine,
K2S2O8, CuSO4, H2O/AcCN, 80 °C, 1,5 h; (vi) NaClO2, NaH2PO4;
t-BuOH/THF, room temp, 24 h).
Figure 3. Synthesis of 2,6-dimethylaminonaphthalene: (i) TrNH2,
AcCN, 50 °C, 72 h; (ii) TFA, DCM, room temp, 35 min; (iii) MeOH,
room temp, 2 h; (iv) 1 M HCl in MeOH.
with tritylamine, followed by acidic solvolysis of the tritylated
amine (7) (Figure 3).32
Several other commercially available diamines were used as
linkers for the reaction with the carboxylic derivatives. Butyl-
amine was used for generating reference monomeric compound
10 (Figure 4), and benzylamine was used for generating
compound 12, both containing a uracil moiety with a carboxa-
mide group at C5 (Figure 4).
The first series of derivatives, containing either a methyl or
an ethoxycarbonylmethyl group at N1, was synthesized using
reaction with thionyl chloride to generate the corresponding acyl
chloride, followed by reaction with the corresponding amine in
pyridine (Figure 4a).
Since the yields obtained with this method were not optimal
(25-30%), mainly because of loss of product during workup,
a second series of derivatives was obtained by activation of the
carboxylic moiety of 5 with fluoride using 2,4,6-trifluoro-1,3,5-
triazine as fluorinating agent, thus providing the stable inter-
mediate 11 which could be isolated. Subsequent reaction of the
acyl fluoride 11 with the corresponding diamine or monoamine
in acetonitrile gave the compounds 12-17 (Figure 4b). Yields
were in the range 37-66%, with lowest value for the short,
rigid m-xylylene bridge.
Screening of Antiproliferative and Differentiation
Inducing Properties. We first determined for all the synthesized
molecules the effects on cell proliferation. To this aim, K562
cells were cultured in the presence of increasing concentrations
of compounds and cell number per milliliter was determined
Conclusions
In the present paper we have demonstrated for the first time
that uracil derivatives bearing a N1-alkyl chain and a 5-car-

90 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Accetta et al.
Figure 4. Synthesis of monomeric and dimeric uracil derivatives: (a) 12 and 13 via acyl chloride ((i) SOCl2, DMF, 70-80 °C, 2 h; (ii) (H2N)n-G,
Py, 2 h); (b) 19-23 via acyl fluoride ((iii) H2N-G-NH2 or H2N-G-NH2. 2HCl, DIEA, AcCN, 80 °C, 7 h; (iv) 1 M HCl, 0.5 h).
Figure 5. Effects of compounds 9 and 14 on in vitro proliferation (A and C) and erythroid differentiation (B and D) of K562 cells. Cells were
cultured in the absence (O) or in the presence of increasing amounts (b, 400 µM; 9, 600 µM; 2, 800 µM) of compound 9 (A and B) and compound
14 (C and D). After the indicated length of time, cell number per milliliter and the proportion of benzidine-positive cells were determined. Representative
images of benzidine staining assay of 6 days of K562 cell cultures treated with compounds 9 and 14 (600 µM) are shown in the relative insets. The
results of three independent experiments are reported in Table 1.
boxamide linker can be considered to be a new class of erythroid
differentiation inducers, and that dimeric derivatives with
suitable spacers have the best performing characteristics: low
cytoxicity and higher differentiating ability. The dependence
of the induction of increase of hemoglobin-containing cells upon
both the length and rigidity of the linking moiety suggests a
cooperative mechanism. Furthermore, the best results were
obtained with the compound bearing a naphthalene linker, which
avoids collapse of the uracil moieties, indicating that a possible
recognition of complementary functionalities (such as adenine
derivatives) could be implicated in the induction of biological
properties. Further studies are needed to evaluate the biological
mechanisms implicated in this process.
These findings can be the starting point for the quest for more
effective and specific drugs for the induction of terminal
erythroid differentiation, ultimately leading to new insights in
the treatment of neoplastic diseases with molecules acting by
inducing differentiation rather than by exerting cytotoxic effects.
In addition, these molecules might be of interest for the
experimental treatment of ꢀ-thalassemic erythroid cells for
which the induction of γ-globin mRNA could be very
beneficial.19,21 In this respect it has been demonstrated that

Uracil Dimers for Erythroid Differentiation
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 91
algorithm with SCF convergence of 1.5936 × 10-13 au for energy,
Table 1. Antiproliferative Effects (IC50) and Percentage of
Benzidine-Positive Cells after Treatment with the Various Compounds
and Concentration Useda
and the STO 3G as the basis set.31
Synthetic Procedures. Methyl 2,4-Dioxo-1,2,3,4-tetrahydropy-
rimidine-5-carboxylate (1). A round-bottomed flask containing 50
mL of MeOH was cooled at 10 °C, and SOCl2 (9.62 mmol, 0.70
mL) was added carefully. After 5 min isoorotic acid was added
and the mixture was heated and refluxed for 18 h. Then the solvent
was removed, and the white powder obtained was washed many
times with MeOH, suspended in hexane, and filtered to yield 1.10 g
of 1 (yield >99%). Dec T > 250 °C. 1H NMR (300 MHz, DMSO-
d6, 25 °C) δ (ppm) 3.68 (s, 3H), 8.13 (s, 1H), 11.31 (s, 1H), 11.60
(s, 1H). 13C NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 50.6, 99.8,
155.2, 157.1, 161.7, 164.5. FT-IR (KBr pellet) ν (cm-1) 1781, 1738,
1620. MS (ESI+): m/z [MH+] 171.0, [MNa+] 192.9. Anal.
(C6H6N2O4) H. C: calcd, 42.36; found, 41.77. N: calcd, 16.47;
found, 15.64.
1-Methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic Acid
(2). Isoorotic acid (1.83 mmol, 300 mg) was suspended in
hexamethydisilazane (HMDS) (11.50 mmol, 2.5 mL), and trim-
ethylchlorosilane (TMS-Cl) (1.10 mmol, 0.10 mL) was added. The
mixture was refluxed in a closed tube at 120 °C for 4 h (until the
mixture appeared colorless). The reaction temperature was raised
to room temperature, and then iodomethane (30.0 mmol, 1.85 mL)
was added. The mixture was then heated to 50 °C and kept
overnight at the same temperature. The solvent was then evaporated,
and the residue was stirred with 3 mL of ice water and 3 mL of
glacial acetic acid for 20 min. The precipitate formed was collected
by filtration and washed with cold water and ethyl acetate to afford
246 mg of a pale-yellow solid corresponding to the product (yield
) 79%). Mp 258.6-259.2 °C. 1H NMR (300 MHz, DMSO-d6, 25
°C) δ (ppm) 3.38 (s, 3H), 8.67 (s, 1H), 12.19 (s, 1H), 12.67 (s,
1H). 13C NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 36.2, 100.7,
150.0, 154.2, 163.3, 164.8. FT-IR (KBr pellet) ν (cm-1) 1752, 1728,
1704, 1618. MS (ESI+): m/z [MH+] 170.0, [MK+] 209.5. Anal.
(C6H6N2O4) N. C: calcd, 42.36; found, 39.61. H: calcd, 3.55; found,
4.01.
antiproliferative
effect IC50 (µM)
% of benzidine positive
cells after 6 days
concentration
compd
(µM)
1
5
9
10
12
13
14
15
16
17
>800
247 ( 33
>800
536 ( 45
75 ( 7.3
247 ( 23
517 ( 63
600 ( 85
220 ( 35
420 ( 93
5 ( 3.4
30 ( 5.8
1 ( 0.8
800
300
800
800
100
200
600
600
600
600
5 ( 2.3
5 ( 3.3
40 ( 8.4
78 ( 7.3
40 ( 5.5
50 ( 6.8
45 ( 3.5
a Results are presented as average ( SD of three independent experiments
performed. The IC50 was calculated as the concentration of compounds
necessary to decrease cell number (after 4 days of culture period) at 50%
of the values obtained in control untreated K562 cell cultures. The % of
benzidine-positive (hemoglobin-containing) cells was determined after 6
days of induction period at concentrations of the tested compounds indicated
in the rightmost column.
Table 2. Effects of Compound 14 on in Vitro Growth and Erythroid
Differentiation of Human Leukemic K562 Cells
erythroid inductiona
compd
concentration
(% of benzidine-positive cells)
14
Ara-C
mithramycin
rapamycin
butyric acid
600 µM
500 nM
100 nM
1.0 mM
2.0 mM
78 ( 7.3
78.3 ( 4.5
86.8 ( 8.3
75.5 ( 7.5
32.5 ( 3.4
a Results are presented as average ( SD (three independent experiments
performed) of the % benzidine-positive (hemoglobin-containing) cells after
6 days of induction period at the indicated concentrations of the tested
compounds.
Table 3. Effects of Compound 14 on in Vitro Growth of Human Cell
Lines
1-(2-Ethoxy-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylic Acid (3). Compound 3 was synthesized as described
above for compound 2, using ethyl bromoacetate as alkylating agent
(yield ) 75%). Mp 184.2-187.2 °C. 1H NMR (300 MHz, DMSO-
d6, 25 °C) δ (ppm) 1.21 (t, 3H, J ) 7.3 Hz), 4.16 (q, 2H, J ) 7.3
Hz), 4.69 (s, 2H), 8.71 (s, 1H), 12.24 (s, 1H), 12.67 (s, 1H). 13C
NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 13.8, 49.2, 61.3, 102.2,
149.7, 153.5, 163.1, 163.3, 167.4. FT-IR (KBr pellet) ν (cm-1) 1793,
1740, 1712, 1625. MS (ESI+): m/z [MH+] 243.0, [MNa+] 265.0.
Anal. (C9H10N2O6) C: calcd, 44.63; found, 43.91. H: calcd, 4.16;
found, 4.90. N: calcd, 11.57; found, 10.54.
antiproliferative
cell line
phenotype/origin
effecta (IC50, µM)
K562
RD
MDA
MCF7
IB3-1
chronic myelogenous leukemia (CML)
rhabdomyosarcoma
breast cancer
breast cancer
bronchial epithelial cell line (cystic fibrosis)
517 ( 63
76.6 ( 9.3
171.2 ( 27.5
197.4 ( 32.4
505 ( 58
a Results are presented as average ( SD (three independent experiments
performed) of concentration needed to obtain 50% inhibition of cell growth
after a 4 day culture period.
5-Methyl-1-octylpyrimidine-2,4(1H,3H)-dione (4). Compound 4
was synthesized according to a literature procedure.38 Mp
inducers of K562 erythroid differentiation are often able to
induce fetal hemoglobin production in erythroid cells isolated
from ꢀ-thalassemia patients.19
1
111.9-113.5 °C (yield ) 22%). H NMR (300 MHz, CDCl3, 25
°C) δ (ppm) 0.82 (t, 3H, J ) 6.9 Hz), 1.21-1.25 (m, 10H), 1.61
(q, 2H, J ) 6.5 Hz), 3.64 (t, 2H, J ) 6.5 Hz), 6.96 (s, 1H), 10.09
(s, 1H). 13C NMR (75 MHz, CDCl3, 25 °C) δ (ppm) 12.2, 13.9,
22.5, 26.3, 29.0, 29.1, 31.6, 48.4, 110.4, 140.4, 151.1, 164.8. FT-
IR (KBr pellet) ν (cm-1) 3160, 3067, 3029, 2955, 2926, 2854, 1692,
1653. MS (ESI+): m/z [MH+] 239.2, [MNa+] 261.3, [MK+] 277.2.
Anal. (C13H22N2O2) C, H, N.
Experimental Section
Chemicals were purchased from Aldrich, Fluka, Acros, or Merck
and have been used without further purification. TLC was performed
on aluminum sheets coated with silca gel F254, 0.2 mm. Flash
chromatography was carried out under nitrogen pressure using
Merck silica gel 60H. NMR spectra were measured on Brucker
AC 300 and on a Varian INOVA 600 instruments. Melting points
were determined on Gallenkamp melting apparatus and are uncor-
rected. FT-IR spectra were recorded on Nicolet FT-IR. Mass spectra
were recorded on Waters Acquity mass spectrometer.
Molecular Mechanics Calculations. Starting structures were
generated using the sketch module of ArgusLAB. Geometry
optimization was carried out using UFF (universal force field).
Electrostatic terms were treated with simple cutoff (10-8 Å), and
structures were optimized using the BFGS (Broyden-Fletcher-
Golfarb-Shanno) algorithm.
1,2,3,4-Tetrahydro-1-octyl-2,4-dioxopyrimidine-5-carboxylic Acid
(5). A solution of 4 (2.55 g, 10.2 mmol) and 2,6-lutidine (4.3 mL)
in acetonitrile (40 mL) was added to a stirring solution of K2S2O8
(5.75 g, 21 mmol) and CuSO4 (0.66 g, 4.2 mmol) in water (40
mL). The mixture was heated at 80 °C for 2 h. Then the mixture
was dried, and the residue was partitioned between ethyl acetate
and saturated aqueous solution of EDTA. The ethyl acetate was
removed from the organic extract, and the resulting yellow oil was
dissolved in 100 mL of t-BuOH-THF-i-butene (6:3:1) mixture.
To this mixture a solution of NaClO2 (12 g, 105 mmol) and
Na2H2PO4 monohydrate (7.5 g, 52.5 mmoles) in water (25 mL)
was added dropwise over a period of 30 min. Then the mixture
was stirred at room temperature overnight. The solvent was removed
and the residue partitioned between saturated aqueous KHSO4 and
Semiempirical Calculations. Geometry optimization was carried
out using the AM1 (NDDO) module of ArgusLAB, the BFGS

92 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
Accetta et al.
ethyl acetate. The organic phase was evaporated, and the residue
was partitioned between methylene chloride and aqueous NaOH
(2 M). The aqueous extract was neutralized with concentrated HCl
at pH 3, and a pale-yellow solid precipitated. The solid was collected
by filtration, washed with abundant cold water, and recrystallized
from acetone-water to obtain 1.54 g (83%) of desired product.
Mp 127.2-128.1 °C. 1H NMR (300 MHz, CDCl3, 25 °C) δ (ppm)
0.88 (t, 3H, J ) 6.9 Hz), 1.26-1.33 (m, 10H), 1.70-1.77 (m, 2H),
3.88 (t, 2H, J ) 7.7 Hz), 8.46 (s, 1H), 9.40 (s, 1H), 12.16 (s-broad,
1H). 13C NMR (75 MHz, CDCl3, 25 °C) δ (ppm) 14.0, 22.5, 26.2,
28.9, 31.6, 50.5, 102.0, 149.0, 152.7, 162.9, 165.0. IR (KBr
pellet) ν (cm-1) 3461, 3420, 3179, 3056, 2956, 2924, 2855, 1744,
1701, 1668. MS (ESI+): m/z [MNa+] 291.2. Anal. (C13H20N2O4)
C, H, N.
and 6 mL of dry pyridine were added. After 2 h the solvent was
removed under reduced pressure and the resulting yellow oil was
suspended in 4 mL of water and acidified to pH 2. The precipitate
collected by filtration was washed with NaOH (1 M), abundant
water, MeOH, and diethyl ether to afford 157 mg of a 10 as a
1
white solid (yield ) 30%). Mp 244.2-245.2 °C. H NMR (300
MHz, DMSO-d6, 25 °C) δ (ppm) 0.88 (t, 3H, J ) 7.2 Hz), 1.29
(m, 2H, J ) 6.9 Hz), 1.45 (m, 2H, J ) 6.9 Hz), 3.25 (q, 2H, J )
6.3 Hz), 3.36 (s, 3H), 8.46 (s, 1H), 8.72 (t, 1H, J ) 5.6 Hz), 11.80
(s, 1H). 13C NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 13.5, 19.4,
31.1, 35.9, 37.8, 104.1, 150.3, 151.6, 161.5, 163.7. FT-IR (KBr
pellet) ν (cm-1) 3441, 3306, 3170, 3047, 2951, 2874, 2835, 1738,
1683, 1615. MS (ESI+): m/z [MH+] 226.2. Anal. (C10H15N3O3) C,
H, N.
1,2,3,4-Tetrahydro-1-octyl-2,4-dioxopyrimidine-5-carbonyl Fluo-
ride (11). To a solution of compound 5 (1.5 g, 5.57 mmol) in 30
mL of dry dichloromethane, cyanuric fluoride (2.9 mL, 33.42 mmol)
was added dropwise, followed by addition of 0.6 mL of dry
pyridine. The mixture was kept under argon and stirred at room
temperature overnight. The reaction mixture was extracted with
water, and the organic layer was dried over Na2SO4 and filtered on
a sintered glass (G5) funnel. The solvent was removed to yield a
yellow-brown solid. Recrystallization from chloroform-hexane
N,N′-[Naphthalene-2,7-diyldi(methylene)]bis(1,1,1-triphenyl-
methanamine) (7). 2,7-Dibromonaphthalene (1.92 mmol, 610 mg)
and tritylamine (7.71 mmol, 2.0 g) were dissolved in 30 mL of
acetonitrile and heated at 50 °C for 96 h. Then the solvent was
removed and the residue was partitioned between saturated NaHCO3
and DCM. The organic layer was dried with Na2SO4 and filtered,
and the solvent was evaporated. The solid obtained was purified
byflashchromatographyonsilicagelusingahexane-dichloromethane
6/4 mixture to afford 990 mg of 7 as a white solid (yield ) 79%).
Mp 221.6-223.3 °C. 1H NMR (300 MHz, CDCl3, 25 °C) δ (ppm)
1.28 (s, 2H), 3.51 (s, 4H), 7.21-7.26 (m, 6H), 7.31-7.36 (m, 12H),
7.46 (dd, 2H, J1 ) 8.4 Hz, J2 ) 1.3 Hz), 7.61-7.64 (m, 12H),
7.79 (d, 2H, J ) 8.4 Hz), 7.88 (d, 2H, J ) 1.3 Hz). 13C NMR (75
MHz, CDCl3, 25 °C) δ (ppm) 48.0, 71.0, 125.7, 126.2, 126.3, 126.3,
127.6, 127.9, 128.6, 131.7, 133.5, 138.6, 146.0. FT-IR (KBr pellet)
ν (cm-1) 3315, 3057, 3019, 2850, 1488, 1448. MS (ESI+): m/z
[M-2Tr-NH3+] 170.1, [Tr+] 243.2. Anal. (C50H42N2) H, N. C: calcd,
89.51; found, 88.85.
1
afforded 1.12 g (75%) of 11. Mp 125.6-127.3 °C. H NMR (300
MHz, CDCl3, 25 °C) δ (ppm) 0.87 (t, 3H, J ) 6.9 Hz), 1.27-1.39
(m, 10H), 1.72-1.76 (m, 2H), 3.86 (t, 2H, J ) 7.2 Hz), 8.28 (s,1H),
9.14 (s, 1H). 13C NMR (75 MHz, CDCl3, 25 °C) δ (ppm) 14.0,
2
22.5, 26.2, 28.9, 29.0, 29.6, 31.6, 50.5, 99.5 (d, JCF ) 61.6 Hz),
149.0, 152.1 (d, 1JCF ) 333.0 Hz), 154.6, 158.2. FT-IR (KBr pellet)
ν (cm-1) 3447, 3182, 3053, 2959, 2923, 2854, 1840, 1820, 1800,
1726, 1696, 1619. MS (ESI+): m/z [MNa+] 293.2, [MK+] 309.2.
Anal. (C13H19FN2O3) H. C: calcd, 57.77; found, 56.85. N: calcd,
10.36; found, 11.37.
Naphthalene-2,7-diyldimethanamine Hydrochloride (8). Com-
pound 7 (1.48 mmol, 960 mg) was dissolved in a mixture of 10
mL of TFA and 7 mL of DCM (an intense yellow color appears
immediately after the addition of TFA), and the mixture was stirred
at room temperature for 40 min. Then 10 mL of MeOH was added
and the mixture was stirred for 1 h until it became colorless. The
solvent mixture obtained was evaporated, and the resulting oil was
dissolved in a minimum amount of 2 M HCl in MeOH and stored
at -20 °C for 1 h. A white precipitate appeared, which was
separated by centrifugation and washed with diethyl ether to afford
365 mg of the corresponding diamine hydrochloride (yield ) 96%).
General Procedure for Compounds 12-17. A solution of 11
(0.37 mmol) and DIEA (1.85 mmol) in acetonitrile (6 mL) was
stirred at room temperature for 5 min. Then the diamine or the
corresponding hydrochloride (0.185 mmoles) was added and the
mixture was refluxed for 7 h. The reaction mixture was poured
into 20 mL of 1 M aqueous HCl, and a yellow-brown precipitate
appeared. The solid was collected by filtration and washed with
water to afford the product.
N-Benzyl-1-octyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxamide (12). Compound 12 was synthesized according the general
procedure described above using an 10-fold excess of benzylamine
(yield ) 88%). Mp 180.3-181.6 °C. 1H NMR (300 MHz, DMSO-
d6, 25 °C) δ (ppm) 0.85 (t, 3H, J ) 6.3 Hz), 1.21-1.29 (m, 10H),
1.56-1.60 (m, 2H), 3.80 (t, 2H, J ) 7.2 Hz), 4.48 (d, 2H, J ) 6.0
Hz), 7.24-7.35 (m, 5H), 8.50 (s, 1H), 9.13 (t, 1H, J ) 6.0 Hz),
11.83 (s, 1H). 13C NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 13.9,
22.0, 25.6, 28.3, 28.5, 31.1, 42.0, 48.4, 99.5, 104.4, 126.8, 127.2,
128.3, 139.2, 150.0, 151.0, 161.7, 163.5. FT-IR (KBr pellet) ν
(cm-1) 3302, 3169, 3115, 3045, 2955, 2922, 2853, 1727, 1678,
1632, 1607. MS (ESI+): m/z [MH+] 358.3, [M2H+] 715.6. Anal.
(C20H27N3O3) H, N. C: calcd, 67.20; found, 65.47.
N,N′-Phenylene-1,3-diylbismethylene(1-octyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide) (13). Yield ) 37%. Dec T
> 210 °C. 1H NMR (300 MHz, DMSO-d6, 25 °C) δ (ppm) 0.85 (t,
6H, J ) 6.6 Hz), 1.19-1.30 (m, 20H), 1.55-1.64 (m, 4H), 3.81
(t, 4H, J ) 7.5 Hz), 4.46 (d, 2H, 6.0 Hz), 7.16-7.31 (m, 4H), 8.50
(s, 2H), 9.12 (t, 2H, J ) 6 Hz), 11.84 (s, 2H). 13C NMR (75 MHz,
DMSO-d6, 25 °C) δ (ppm) 13.9, 22.0, 25.6, 28.3, 28.5, 31.1, 42.0,
48.42, 104.3, 125.7, 128.5, 139.3, 149.7, 151.1, 153.4, 161.7, 163.1.
FT-IR (KBr pellet) ν (cm-1) 3299, 3235, 3015, 2956, 2922, 2852,
2813, 1731, 1693, 1637, 1608. HRMS (MALDI+): m/z [M + Na+]
calcd for C34H48N6NaO6+, 659.3533; found 659.3961. Anal.
(C34H48N6O6) H, N. C: calcd, 64.13; found, 62.42.
N,N′-Naphthalene-2,7-diylbismethylene(1-octyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxamide) (14). Yield ) 66%). Dec T
> 280 °C. 1H NMR (300 MHz, DMSO-d6, 25 °C) δ (ppm) 0.85 (t,
6H, J ) 6.9 Hz), 1.17-1.30 (m, 20H), 1.55-1.64 (m, 4H), 3.81
(t, 4H, J ) 6.6 Hz), 4.64 (d, 2H, 5.4 Hz), 7.42 (d, 2H, J ) 8.4 Hz),
1
Dec T > 285 °C. H NMR (300 MHz, D2O, 25 °C) δ (ppm) 4.33
(s, 4H), 7.59 (dd, 2H, J1 ) 8.5 Hz, J2 ) 1.7 Hz), 7.98 (d, 2H, J )
1.7 Hz), 8.02 (d, 2H, J ) 8.4 Hz). 13C NMR (75 MHz, D2O, 25
°C) δ (ppm) 46.5, 130.2, 131.6, 132.3, 134.5, 136.1, 136.2. FT-IR
(KBr pellet) ν (cm-1) 3125, 3033, 3013, 2997, 2965, 2867, 1599,
1495, 1480. MS (ESI+): m/z [MH+ - NH3] 170.0, [MH+] 187.1.
Anal. (free amine) (C12H14N2) H, N. C: calcd, 77.38; found, 76.77.
Diethyl 2,2′-{Oxybis[ethane-2,1-diyliminocarbonyl(2,4-dioxo-3,4-
dihydropyrimidine-5,1-diyl)]}diacetate (9). Dry DMF (3 mL),
compound 3 (0.88 mmol, 150 mg), and SOCl2 (1.80 mmol, 130
µL) were heated to 80 °C for 2 h under N2 atmosphere. Then
2-(aminoethoxy)ethanamine (0.44 mmol, 45 mg) and 3 mL of dry
pyridine were added. After 2 h the solvent was removed under
reduced pressure and the resulting oil was suspended in 10 mL of
water. A yellow, pale precipitate appeared and was collected and
washed with methanol and ethyl ether to afford 45 mg of 9 (yield
) 25%). Dec T > 285 °C. 1H NMR (300 MHz, DMSO-d6, 25 °C)
δ (ppm) 3.35 (s, 6H), 3.41 (q, 4H, J ) 6.0 Hz), 3.50 (t, 4H, J )
6.0 Hz), 8.45 (s, 2H), 8.84 (t, 2H, J ) 6.0 Hz), 11.77 (s, 2H). 13C
NMR (75 MHz, DMSO-d6, 25 °C) δ (ppm) 35.9, 38.2, 67.7, 104.0,
150.3, 151.6, 161.7, 163.5. FT-IR (KBr pellet) ν (cm-1) 1730, 1696,
1611, 1636. MS (ESI+): m/z [MH+] 409.1, [MNa+] 431.1. Anal.
(C22H28N6O11) H. C: calcd, 47.83; found, 45.92. N: calcd, 15.21;
found, 14.79.
N-Butyl-1-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-car-
boxamide (10). Dry DMF (3 mL), compound 2 (2.35 mmol, 400
mg), and SOCl2 (4.70 mmol, 341 µL) were heated to 80 °C for 2 h
under N2 atmosphere, and then butylamine (4.70 mmol, 470 µL)

Uracil Dimers for Erythroid Differentiation
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1 93
7.71 (s, 2H), 7.85 (d, 2H, J ) 8.4 Hz), 9.22 (t, 2H J ) 5.4 Hz),
11.85 (s, 2H). 13C NMR (150 MHz, DMSO-d6, 90 °C) δ (ppm)
13.1, 21.3, 25.1, 27.77, 27.79, 27.84, 41.9, 48.0, 104.4, 124.9, 125.2,
127.2, 130.9, 132.5, 136.6, 150.3, 157.6, 158.9, 161.4. FT-IR (KBr
pellet) ν (cm-1) 3297, 3128, 3010, 2956, 2920, 2851, 2809, 1728,
1696, 1636, 1608. HRMS (MALDI+): m/z [M + Na+] calcd for
C38H50N6NaO6 709.3690, found 709.4341. Anal. (C38H50N6O6 ·H2O)
C, H, N.
Thalassemia Research Network) for support. Valentina Mileo
(Chiesi Farmaceutici) is gratefully acknowledged for performing
the HRMS experiments.
Supporting Information Available: Full combustion data
analysis for compounds 1-5 and 7-17, 1D NOESY of N1
substituted compound 3, and graphs of cell proliferation and
differentiation for compound 12. This material is available free of
charge via the Internet at http://pubs.acs.org.
N,N′-Hexane-1,6-diylbis(1-octyl-2,4-dioxo-1,2,3,4-tetrahydropy-
rimidine-5-carboxamide) (15). Yield ) 58%. Dec T > 237 °C. 1H
NMR (300 MHz, DMSO-d6, 25 °C) δ (ppm) 0.84 (t, 6H, J ) 6.5
Hz), 1.20-1.28 (m, 24H), 1.42-1.49 (m, 4H), 1.54-1.61 (m, 4H),
3.25 (m, 4H, J ) 5.7 Hz), 3.80 (t, 4H, J ) 7.1 Hz), 8.45 (s, 2H),
8.74 (t, 2H, J ) 5.7 Hz), 11.83 (s, 2H). 13C NMR (75 MHz, DMSO-
d6, 25 °C) δ (ppm) 13.8, 21.9, 25.5, 25.9, 28.2, 28.4, 28.8, 28.9,
31.0, 38.1, 48.3, 104.4, 149.9, 150.6, 161.4, 163.5. FT-IR (KBr
pellet) ν (cm-1) 3298, 3241, 3013, 2956, 2921, 2853, 2813, 1729,
1697, 1635, 1608. HRMS (ESI+): m/z [M + Na+] calcd for
C32H52N6NaO6+, 639.3846; found, 639.3835. Anal. (C32H52N6O6)
H, N. C: calcd, 62.31; found, 61.28.
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6.8 Hz), 1.21-1.28 (m, 28H), 1.42-1.50 (m, 4H), 1.54-1.60 (m,
4H), 3.23 (m, 4H, J ) 6.1 Hz), 3.80 (t, 4H, J ) 6.8 Hz), 8.45 (s,
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Acknowledgment. This work was partially supported by a
grant from MIUR (PRIN2005 Grant No. 2005038704). R.G. is
supported by Associazione Italiana Ricerca sul Cancro (AIRC),
Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo),
Associazione Veneta per la Lotta alla Talassemia, Rovigo, Italy
(AVLT), Telethon (Grant GGP07257), and STAMINA Project.
We thank the EU Project ITHANET (eInfrasctructure for

94 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 1
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