Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones in human cancer cells

Article abstract of DOI:10.1016/j.ejmech.2015.06.040

Abstract The synthesis of a series of 14 new 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones from the 1,3-dipolar cycloaddition reaction of 1-allyl-4-(trihalomethyl)pyrimidin-2(1H)-ones with aryl nitrile oxides is described. Also, the antiproliferative activity of the title compounds was tested against five human tumoral cell lines: MCF-7 breast cancer cell line, ER+ (estrogen receptor positive); HepG-2 (hepatoma); T-24 (bladder cancer); HCT-116 cell (colorectal carcinoma); and CACO-2. The preliminary results are promising, since three compounds presented IC₅₀ values below 2 μM, as well as moderate to high selectivity.

Full text of DOI:10.1016/j.ejmech.2015.06.040

Accepted Manuscript
Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-
dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones in human cancer
cells
Marcio M. Lobo, Cassiana M. Viau, Josiane M. dos Santos, Helio G. Bonacorso,
Marcos A.P. Martins, Simone S. Amaral, Jenifer Saffi, Nilo Zanatta
PII:
S0223-5234(15)30115-X
10.1016/j.ejmech.2015.06.040
EJMECH 7967
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 April 2015
Revised Date: 22 May 2015
Accepted Date: 19 June 2015
Please cite this article as: M.M. Lobo, C.M. Viau, J.M. dos Santos, H.G. Bonacorso, M.A.P. Martins, S.S.
Amaral, J. Saffi, N. Zanatta, Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-
dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones in human cancer cells, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.040.
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ACCEPTED MANUSCRIPT
GRAPHICALABSTRACT
Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-
dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones
in
human
cancer cells
a,
b,
a
a
Marcio M. Lobo, Cassiana M. Viau, Josiane M. dos Santos, Helio G. Bonacorso, Marcos A. P.
a
b
b*
a*
Martins, Simone S. Amaral, Jenifer Saffi, Nilo Zanatta
a)
Núcleo de Química de Heterociclos (NUQUIMHE), Departamento de Química, Universidade Federal de Santa
Maria, 97105-900, Santa Maria, Brazil, Fax: +55 55 3220 8756. Email: nilo.zanatta@ufsm.br
b)
Laboratório de Genética Toxicológica, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento
Leite, 245, Anexo II, 90050-170 – Porto Alegre, RS, Fax: +55 51 3303 8861. Email: jenifers@ufcspa.edu.br

ACCEPTED MANUSCRIPT
Synthesis and cytotoxic activity evaluation of some novel 1-(3-(aryl-4,5-
dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones
in human
cancer cells
a,†
b,†
a
a
Marcio M. Lobo, Cassiana M. Viau, Josiane M. dos Santos, Helio G. Bonacorso,
a
b
b*
a*
Marcos A. P. Martins, Simone S. Amaral, Jenifer Saffi, Nilo Zanatta
a)
Núcleo de Química de Heterociclos (NUQUIMHE), Departamento de Química,
Universidade Federal de Santa Maria, 97105-900, Santa Maria, Brazil, Fax: +55 55
3
220 8756. Email: nilo.zanatta@ufsm.br
b)
Laboratório de Genética Toxicológica, Universidade Federal de Ciências da Saúde de
Porto Alegre, Rua Sarmento Leite, 245, Anexo II, 90050-170 – Porto Alegre, RS, Fax:
+55 51 3303 8861. Email: jenifers@ufcspa.edu.br
†
These authors contributed equally to this work.
Abstract
The synthesis of a series of 14 new 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones from the 1,3-dipolar cycloaddition reaction of 1-
allyl-4-(trihalomethyl)pyrimidin-2(1H)-ones with aryl nitrile oxides is described. Also,
the antiproliferative activity of the title compounds was tested against five human
tumoral cell lines: MCF-7 breast cancer cell line, ER+ (estrogen receptor positive);
HepG-2 (hepatoma); T-24 (bladder cancer); HCT-116 cell (colorectal carcinoma); and
CACO-2. The preliminary results are promising, since three compounds presented IC50
values below 2 ꢀM, as well as moderate to high selectivity.
1

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Keywords: 4,5-dihydroisoxazoles; pyrimidin-2-ones; nitrile oxides; antiproliferative
activity; human tumoral cell
1
. Introduction
Cancer is a complex disease that involves molecular, cellular, and tissual [1] factors
and, in most cases, is caused by continuous exposure to mutagens and carcinogens such
as sunlight, toxic chemicals, ionizing energies, etc. [2]. Worldwide, there were 14.1
million new cancer cases, 8.2 million cancer deaths, and 32.6 million people living with
cancer (with 5 years or less of diagnosis) in 2012. Fifty-seven percent (8 million) of
new cancer cases, 65 % (5.3 million) of the cancer deaths, and 48 % (15.6 million) of
the cancer cases with 5 years or less of diagnosis occurred in less developed regions [3].
In general, antineoplastic agents act primarily in the cell division process and affect both
normal and abnormal cells [4]. Consequently, there are often severe side effects
associated with their administration. The most common side effects are alopecia, dry
mouth, dyspepsia, hematological disorders, and fatigue [5,6].
In order to overcome such drawbacks and, ultimately, to improve patients’ health and
wellness, several studies are being conducted to develop new anticancer agents that
have superior activity and/or have fewer side effects than the currently used drugs [7].
In this context, pyrimidine nucleosides and their derivatives are considered to be an
important molecular template in the development of new and improved antiproliferative
agents. Several structural changes in both the nitrogenated base and the furanose ring
have been proposed, with the aim of developing leading compounds or enhancing the
antitumor activity of existing ones.
2

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As a result of these studies, a series of DNA bases and nucleoside analogues have been
developed and have been used in various types of cancer treatment. Among these, one
can highlight 5-fluorouracil, 6-mercaptopurine and cytarabine (Figure 1) [8-10].
Figure 1
Unfortunately, these drugs present a wide range of unwanted side effects and limited
efficacy. Thus, the development of new, more effective and less toxic agents remains an
ongoing challenge.
Since structural modifications of a parent compound or a class of compounds are often
used to improve drug candidates, we envisioned synthesizing a series of trihalomethyl-
pyrimidine sugar-modified nucleoside derivatives and evaluating their cytotoxic effect
on several cancer cell lines. The central idea was: i) to replace the pentose ring with a 4-
5
-dihydroisoxazole in order to improve the stability in physiological pH; ii) to use a
methylenic linkage between the pyrimidinone and the sugar-based heterocycle, thus
preventing the acidic hydrolysis which is often observed in the glycosidic bond; and iii)
to insert a trihalomethyl group into the pyrimidinone moiety, due to the well-known
improvements in lipophilicity and stability, which are often associated with such groups
[
11,12] (Figure 2).
Figure 2
. Results and discussion
2
2
.1. Chemistry
Scheme 1 outlines the preparation of 1-(3-aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones 8-9aꢀg in a convergent and efficient way from the
,3-dipolar [3+2] cycloaddition reaction between N1-allylpyrimidinones 4-5 and nitrile
̶
1
3

ACCEPTED MANUSCRIPT
oxides (7a-g). The N1-allylpyrimidinones 4-5 were obtained from the
cyclocondensation reaction of enones 1-4 with allylurea (3) in methanol and in the
presence of hydrochloric acid [13]. The hydroxymoyl chlorides were prepared from the
reaction of aldoximes with N-chlorosuccinimide (NCS) and hydrochloric acid in THF
and were converted in situ to the corresponding nitrile oxides 7a
trimethylamine [14]. Addition of the N1-allylpyrimidin-2-ones 4-5 to the nitrile oxides
a-g furnished the desired trihalomethyl-pyrimidine sugar-modified nucleoside
analogues 8-9aꢀg at good yields, after a 4 h period under reflux.
̶ꢀ g by using
7
̶
Scheme 1
Although similar synthetic procedures have been applied to access other modified
nucelosides and nucleobases, our proposal brings some interesting improvements
[
13a,15]. The highly regioselective synthesis of 1-allylpyrimidinones 4-5 in a single step
is one of these improvements. This straightforward method, which has already been
reported by our research group, avoids a five-step procedure that would involve the
following: pyrimidinone cyclization with 1,3-dielectrophiles and urea; protection of
pyrimidinone nitrogen atoms; pyrimidinone N-1 deprotection; N1-allylation; and
pyrimidinone N-3 deprotection. Additionally, the N-allylation procedure of our choice is
simpler and more efficient than the Mitsunobu-type conditions previously described
[
16].
Pyrimidinone 4 and oxime 6a were selected for the optimization of reaction conditions
Table 1). The method described by Bujak [17] was adapted for our substrates and we
(
established that the best reaction condition was THF reflux for 4 h (Entry 6, Table 1).
Table 1
4

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Once the best reaction condition has been established, the synthesis of all compounds of
the series is performed. It was observed that neither the oxime aryl substituents nor the
trihalomethyl group of the pyrimidinone significantly influenced the reaction yields.
These results show the method’s versatility and prove that it can be used to access a
collection of sugar-modified nucleoside derivatives similar to compounds 8–9a–g.
Table 2
It has long been established that 1,3-dipolar cycloadditions between nitrile oxides and
alkenes may form two different regioisomers, with each one being a pair of enatiomers
(Figure 3) [18]. The regiochemistry of these reactions has been extensively studied by
Houk and Sustmann, and it essentially depends on steric and electronic effects [19].
Figure 3
1
13
The regiochemistry of the products 8-9 was assigned based on the H and C NMR
1
13
spectra and 2D HMBC experiments. The H and C NMR spectra of the synthesized
products 8-9 show a characteristic chemical shift pattern of 3,5-substituted isoxazolines.
For compound 8d, for example, the isoxazolinic hydrogen H-5’ appears as a multiplet at
δ = 5.12 ppm and the two methylene carbons neighbor to the stereogenic center,
comprising four diastereotopic hydrogens, show four doublets of doublets. Two of these
double doublets, belonging to the hydrogens attached to the exocyclic methylene,
appear at δ = 4.41 and 3.97 ppm and the other two doublets of doublets, at δ = 3.61 and
1
3
3
.28 ppm, for the endocyclic methylene hydrogens. In the C NMR spectrum, the
regiochemistry of the 1,3-dipolar cycloaddition reaction could be confirmed by the
presence of the C-4’ and C-5’ carbons, at δ = 76.7 and 37.7 ppm, respectively [20]. In
the HMBC spectrum, a cross-peak was not observed between the exocyclic H-7 with
the endocyclic carbon C-3’ (4 bonds), but a cross-peak of the endocyclic methylene
2
hydrogens with the sp carbon of the isoxazolinic ring was observed. This also shows
5

ACCEPTED MANUSCRIPT
that the regioisomer obtained is the 3,5-isoxazoline because, if its 3,4-substituted isomer
were obtained, one would observe a cross-peak between H-7 and C-3'. Compound 8d
was also confirmed by x-ray diffraction (Figure 4). All products of the series 8-9a-g
1
13
showed the same H- and C-NMR spectral pattern, which is consistent with 3,5-
disubstituted isoxazolines, as described for compound 8d.
Figure 4
2
.2. Biological studies
The in vitro cytotoxic activity of some trihalomethyl-pyrimidine sugar-modified
nucleoside derivatives against a wide range of cancer cell lines is presented in Table 3.
The antiproliferative effects were tested using the XTT assay and were quantified in
terms of IC values. The lower the IC value, the higher the antiproliferative activity
5
0
50
[
7]. The ratio between the cytotoxic parameters found in HEK-293 non tumor derived
cell line and those observed in tumoral cell lines can be considered as a measurement of
compound selectivity index (SI). SI ratios between 3 and 6 refer to moderate selectivity,
and ratios higher than six indicate high selectivity, whereas compounds that do not
fulfill either of these criteria are rated as non-selective [6,11]. The SI value is also
shown in Table 3. Of the twelve compounds tested, compounds 8a, 8e, and 9c stand out
due to their low IC concentrations (IC < 2 ꢀM) and moderate to high selectivity
5
0
50
(entries 1, 4, and 9, Table 3).
Table 3
Our results suggest that 8a exerted the highest cytotoxicity against HepG-2 cells, with
an IC value of 1.04 ꢀM and inhibition 14 times higher than normal cells (HEK-293).
5
0
More importantly, 8a was non-cytotoxic against HEK-293 non-tumor derived cell lines.
6

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Additionally, 8e and 9c also showed significant inhibitory activity, in human cancer cell
line T-24 and CACO-2, respectively (Table 3).
In comparison with mitoxantrone, 8a, 8e, and 9c demonstrated lower IC values in
50
tumor cell lines. Considering both cytotoxicity and the selectivity index, 8a displayed
the best profile in HepG-2 cells. Such characteristics are very important because
hepatocellular carcinoma (HCC) is a common malignant disease that is often associated
with chemoresistance [12]. Further studies with 8a, using other biological models, are
currently being performed in our laboratories and should provide a better understanding
of the mechanisms underlying these effects in human HCC cell lines.
4
. Conclusion
This study showed an elegant and efficient synthesis of a new series of trihalomethyl-
pyrimidine sugar-modified nucleoside analogues from the 1,3-dipolar [3+2]
cycloaddition reaction of 6-substituted 1-allyl-4-(trihalomethyl)pyrimidin-2(1H)-ones
with different nitrile oxides, derived from the corresponding aryl oximes. The
1
characterization and regiochemistry of the products were assigned based on the H and
1
3C NMR spectra and 2D HMBC experiments, and subsequently confirmed by x-ray
diffraction. In vitro antiproliferative activity of these compounds evaluated against five
human cancer cell lines (MCF-7, HepG-2, T-24, HCT-116, and CACO-2) showed that
three of the compounds (8a, 8e, and 9c) exhibited high antiproliferative activity (IC50
less than 1.40 ꢀM) and appeared to be less toxic against normal cells. In particular, 8a
was three times more selective than MXT standard anticancer drugs. Overall, our results
for the first time highlight the potential of novel 1-(3-(aryl-4,5-dihydroisoxazol-5-
yl)methyl)-4-trihalomethyl-1H-pyrimidin-2-ones obtained from a simple and easy-to-
obtain synthetic procedure. These novel elements could be employed as therapeutic
7

ACCEPTED MANUSCRIPT
drugs for HCC treatment, thus providing the rationale for future experimental use in
humans.
5
. Experimental Protocols
1
All the products described in this article were fully characterized by melting point, H
1
3
and C NMR elemental analysis, and 2D NMR experiments. All melting points were
determined on a Kofler Reichert Thermovar or on a MQAPF-301 apparatus, and are
uncorrected. The CHN microanalyses were performed on a Perkin Elmer 2400
elemental analyzer. High resolution mass spectra were recorded on a Bruker QTOF
1
13
spectrometer in ESI-mode. H- and C-NMR spectra and 2D NMR experiments were
1
13
registered on a Bruker DPX 400 ( H at 400.13 MHz and C at 100.62 MHz) in CDCl
3
or DMSO-d ,using TMS as the internal reference.
6
5
.1. Chemistry
General: The 4-alkoxy-4-substituted-but-3-enones (1–2) were prepared according to
the literature [21]. The N1-allylpyrimidinones 6−9 were obtained from the
cyclocondensation reaction of enones 1–2 with allylurea (3) in methanol and in the
presence of hydrochloric acid, in accordance with previous studies [13]. The oximes
6
a–g were obtained by the reaction of the corresponding aldehyde with hydroxylamine
hydrochloride, in accordance with the method described in the literature [22].
5
4
.1.2. General procedure for the synthesis of 1-(3-aryl-4,5-dihydroisoxazol-5-yl)methyl-
-trihalomethyl-1H-pyrimidin-2-ones (8-9)
To a solution of oximes 6a–g (1.2 mmol) in anhydrous THF (8 mL), N-
Chlorosuccinimide (1.3 mmol, 0.173 g) and hydrochloric acid 37 % (1 mmol, 0.1 mL)
were added and the mixture was stirred at room temperature for 2 h. The pyrimidinones
4
–5 (1.0 mmol) were added to the reaction flask, followed by the dropwise addition of
8

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triethylamine (3.9 mmol, 0.55 mL), and the temperature was raised to reflux for 4 h.
The reaction product was isolated by extraction with dichloromethane (3 x 20 mL) and
the combined organic phases were further extracted with 3 % hydrochloric acid solution
(1 × 20 mL), then dried under anhydrous sodium sulfate, filtered, and finally, the
solvent was evaporated in a rotary evaporator. The products were isolated as stable
solids and they were purified by recrystallization from chloroform or ethanol, in
accordance with the description of the synthesis of each compound.
5
2
.1.2.1. 1-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl) pyrimidin-
1
(1H)-one (8a). Yellow crystal, 98% yield, m.p. 195–197 ºC (ethanol); H NMR (400
MHz, DMSO-d /TMS) δ 8.44 (d, J = 6.9, 1H, H-6), 7.67 – 7.63 (m, 2H, Ar), 7.47 – 7.43
6
(m, 3H, Ar), 7.04 (d, J = 6.9, 1H, H-5), 5.14 – 5.07 (m, 1H, H-5’), 4.25 (dd, J = 13.6,
3
.8, 1H, H-7), 4.10 (dd, J = 13.6, 7.9, 1H, H-7), 3.62 (dd, J = 17.3, 10.6, 1H, H-4’), 3.33
1
3
(
dd, J = 17.3, 6.1, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 170.5 (C-4),
6
1
57.0 (C-3’), 154.2 (C-2), 153.8 (C-6), 130.2, 128.9, 128.7, 126.6 (Ar), 98.5 (C-5), 95.3
(
CCl ), 76.9 (C-5’), 53.3 (C-7), 37.7 (C-4’); Anal. Calcd. for C H Cl N O (371.00):
3
15 12
3
3
2
C, 48.35; H, 3.25, N, 11.28%. Found: C, 48.72; H, 3.23; N, 11.31%.
5
.1.2.2.
1-((3-(4-Fluorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8b). Beige powder, 83% yield, m.p. 205–207 ºC (chloroform); H
NMR (400 MHz, DMSO-d /TMS) δ 8.45 (d, J = 6.9, 1H, H-6), 7.75 – 7.69 (m, 2H, Ar),
6
7
.30 (t, J = 8.9, 2H, Ar), 7.06 (d, J = 6.9, 1H, H-5), 5.14 – 5.06 (m, 1H, H-5’), 4.24 (dd,
J = 13.6, 3.7, 1H, H-7), 4.08 (dd, J = 13.6, 8.1, 1H, H-7), 3.62 (dd, J = 17.3, 10.6, 1H,
1
3
H-4’), 3.39 – 3.35 (m, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 170.6 (C-4),
6
1
63.17 (d, J = 248.1, Ar),156.2 (C-3’), 154.2 (C-2), 154.0 (C-5), 163.2 (d, J = 248.1),
9

ACCEPTED MANUSCRIPT
1
7
2
29.1 (d, J = 8.6), 125.5 (d, J = 3.1), 115.9 (d, J = 22.0) (Ar), 98.6 (C-5), 95.4 (CCl3),
7.1 (C-5’), 53.3 (C-7), 37.8; Anal. Calcd. for C H Cl FN O (388.99): C, 46.12; H,
1
5
11
3
3
2
.84, N, 10.76%. Found: C, 46.27; H, 2.80; N, 11.20%
5
.1.2.3.
1-((3-(2-Toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8c). White powder, 97% yield, m.p. 202–205 ºC (chloroform); H
NMR (400 MHz, CDCl /TMS) δ 8.07 (d, J = 7.0, 1H, H-6), 7.33 – 7.20 (m, 4H, Ar),
3
6
3
6
1
9
.91 (d, J = 6.9, 1H, H-5), 5.14 – 5.05 (m, 1H, H-5’), 4.41 (dd, J = 13.6, 2.5, 1H, H-7),
.97 (dd, J = 13.6, 7.6, 1H, H-7), 3.61 (dd, J = 17.1, 10.6, 1H, H-4’), 3.28 (dd, J = 17.1,
1
3
.2, 1H, H-4’), 2.50 (s, 3H, Me); C NMR (101 MHz, CDCl /TMS) δ 172.1 (C-4),
3
58.2 (C-3’), 155.1 (C-2), 151.5 (C-6), 137.9, 131.7, 129.97, 129.0, 127.6, 126.1 (Ar),
9.8 (C-5), 95.1 (CCl ), 76.6 (C-5’), 53.7 (C-7), 40.7 (C-4’), 22.9 (Me); HRMS-ESI:
3
[
M + H] calcd. for C H Cl N O : 386.0230; found: 386.0227.
16 15 3 3 2
5
.1.2.4.
1-((3-(4-Toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8d). Brown crystal, 80% yield, m.p. 209–211 ºC (ethanol); H
NMR (400 MHz, DMSO-d /TMS) δ 8.46 (d, J = 6.9, 1H, H-6), 7.56 (d, J = 8.0, 2H,
6
Ar), 7.27 (d, J = 8.2, 2H, Ar), 7.06 (d, J = 6.9, 1H, H-5), 5.12 – 5.00 (m, 1H, H-5’), 4.24
(dd, J = 13.5, 3.6, 1H, H-7), 4.06 (dd, J = 13.6, 8.2, 1H, H-7), 3.60 (dd, J = 17.3, 10.7,
1
3
1
H, H-4’), 3.35 – 3.25 (m, 1H, H-4’), 2.34 (s, 3H, Me); C NMR (100 MHz, DMSO-
d6/TMS) δ 170.5 (C-4), 156.8(C-3’), 154.1 (C-2), 153.8 (C-6), 140.0, 129.2, 126.5,
26.1 (Ar), 98.4 (C-5), 95.3 (CCl ), 76.7 (C-5’), 53.2 (C-7), 37.7 (C-4’), 20.8 (Me);
1
3
Anal. Calcd. for C H Cl N O (385.02): C, 49.70; H, 3.65, N, 10.87%. Found: C,
1
6
14
3
3
2
4
9.32; H, 3.71; N, 10.72%
1
0

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5
.1.2.5. 1-((3-(2-Hydroxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8e). Beige powder, 61% yield, m.p. 216–218 ºC (ethanol); H
NMR (400 MHz, DMSO-d /TMS) δ 9.78 (s, 1H, OH), 8.46 (d, J = 6.9, 1H, H-6), 7.44
6
(dd, J = 7.8, 1.5, 1H, Ar), 7.36 – 7.29 (m, 1H, Ar), 7.05 (d, J = 6.9, 1H, H-5), 6.93 (ddd,
J = 15.0, 8.0, 1.6, 2H, Ar), 5.14 – 5.05 (m, 1H, H-5’), 4.26 (dd, J = 13.6, 3.7, 1H, H-7),
4
6
.12 (dd, J = 13.6, 7.9, 1H, H-7), 3.71 (dd, J = 17.6, 10.5, 1H, H-4’), 3.44 (dd, J = 17.6,
1
3
.1, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 170.5 (C-4), 158.0 (Ar), 156.0
6
(
C-3’), 154.1 (C-2), 153.8 (C-6), 131.5, 129.0, 119.3, 116.3, 114.6 (Ar), 98.4 (C-5),
5.3 (CCl ), 76.2 (C-5’), 53.0 (C-7), 39.0 (C-4’); Anal. Calcd. for C H Cl N O
3
9
3
15 12
3
3
(386.99): C, 46.36; H, 3.11, N, 10.81%. Found: C, 46.50; H, 3.00; N, 10.37%
5
.1.2.6. 1-((3-(2-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8f). White powder, 99% yield, m.p. 124–126 ºC (chloroform); H
NMR (400 MHz, DMSO-d /TMS) δ 8.44 (d, J = 6.9, 1H, H-6), 7.51 – 7.40 (m, 2H, Ar),
6
7
.11 (d, J = 8.3, 1H, Ar), 7.06 (d, J = 6.9, 1H, H-5), 6.98 (t, J = 7.5, 1H, Ar), 5.10 – 5.02
(
(
(
(
(
3
m, 1H, H-5’), 4.21 (dd, J = 13.7, 3.6, 1H, H-7), 4.13 (dd, J = 13.6, 7.1, 1H, H-7), 3.83
1
3
s, 3H, OMe), 3.60 (dd, J = 17.8, 10.7, 1H, H-4’), 3.38 – 3.32 (m, 1H. H-4’); C NMR
101 MHz, DMSO-d /TMS) δ 170.5 (C-4), 157.3 (Ar), 156.1 (C-3’), 154.2 (C-2), 154.0
6
C-6), 131.6, 128.9, 120.5, 117.9, 112.1 (Ar), 98.5 (H-5), 95.4 (CCl ), 76.9 (C-5’), 55.7
3
OMe), 53.1 (C-7), 40.0 (C-4’). Anal. Calcd. for C H Cl N O (401.01): C, 47.73; H,
16
14
3
3
3
.50, N, 10.44%. Found: C, 47.85; H, 3.53; N, 10.21%
5
.1.2.7. 1-((3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trichloromethyl)
1
pyrimidin-2(1H)-one (8g). White powder, 94% yield, m.p. 219–221 ºC (chloroform); H
NMR (400 MHz, DMSO-d /TMS) δ 8.45 (d, J = 6.9, 1H, H-6), 7.60 (d, J = 8.9, 2H,
6
1
1

ACCEPTED MANUSCRIPT
Ar), 7.05 (d, J = 6.9, 1H, H-5), 7.01 (d, J = 8.9, 2H, Ar), 5.11 – 4.99 (m, 1H, H-5’), 4.23
(dd, J = 13.5, 3.7, 1H, H-7), 4.05 (dd, J = 13.6, 8.1, 1H, H-7), 3.80 (s, 3H, OMe), 3.58
13
(
dd, J = 17.2, 10.5, 1H, H-4’), 3.30 (dd, J = 17.2, 5.9, 1H, H-4’); C NMR (101 MHz,
DMSO-d /TMS) δ 170.4 (C-4), 160.7 (Ar), 156.4 (C-3’), 154.1 (C-2), 153.8 (C-6),
6
1
28.2, 121.3, 114.13 (Ar), 98.3 (C-5), 95.3 (CCl3), 76.5 (C-5’), 55.2 (OMe), 53.2 (C-7),
7.9 (C-4’); Anal. Cal. for C H Cl N O (401.01): C, 47.73; H, 3.50, N, 10.44%.
3
1
6
14
3
3
3
Found: C, 47.77; H, 3.66; N, 10.55%
5
.1.2.8.
1-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)pyrimidin-
1
2
(1H)-one (9a). White powder, 92% yield, m.p. 181–182 ºC (ethanol); H NMR (400
MHz, DMSO-d /TMS) δ 8.57 (d, J = 6.6, 1H, H-6), 7.67 (dd, J = 6.6, 3.1, 2H, Ar), 7.49
6
–
1
4
3
1
7.43 (m, 3H, Ar), 6.90 (d, J = 6.6, 1H, H-5), 5.16 – 5.06 (m, 1H, H-5’), 4.28 (dd, J =
3.5, 3.8, 1H, H-7), 4.11 (dd, J = 13.5, 8.1, 1H, H-7), 3.62 (dd, J = 17.3, 10.6, 1H, H-
1
3
’), 3.39 – 3.31 (m, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 161.6 (q, J =
6
5.3, C-4), 157.2 (C-3’), 155.5 (C-6), 154.7 (C-2), 130.5, 129.1, 129.0, 126.9 (Ar),
19.7 (q, J = 277.5, CF ), 99.0 (d, J = 1.8, C-5), 77.0 (C-5’), 53.9 (C-7), 37.9 (C-4’);
3
Anal. calcd. for C H F N O (323.09): C, 55.73; H, 3.74; N, 13.00%. Found: C, 55.79;
1
5
12
3
3
2
H, 3.75, N, 12.96%.
5
.1.2.9.
1-((3-(4-Fluorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9b). White powder, 84% yield, m.p. 186–187 ºC (ethanol); H
NMR (400 MHz, DMSO-d /TMS) δ 8.54 (d, J = 6.6, 1H, H-6), 7.72 (dd, J = 8.6, 5.5,
6
2
4
1
H, Ar), 7.28 (t, J = 8.8, 2H, Ar), 6.87 (d, J = 6.6, 1H, H-5), 5.14 – 5.07 (m, 1H, H-5’),
.27 (dd, J = 13.6, 3.9, 1H, H-7), 4.12 (dd, J = 13.5, 7.9, 1H, H-7), 3.61 (dd, J = 17.3,
1
3
0.6, 1H, H-4’), 3.34 (dd, J = 17.3, 6.2, 1H, H-4’); C NMR (101 MHz, DMSO-
1
2

ACCEPTED MANUSCRIPT
d6/TMS) δ 163.1 (d, J = 248.2, Ar), 161.4 (q, J = 35.5, C-4), 156.1 (C-3’), 155.1 (C-6),
1
54.4 (C-2), 128.9 (d, J = 8.7, Ar), 125.44 (d, J = 3.2, Ar), 119.4 (q, J = 277.4, CF3),
15.69 (d, J = 22.0, Ar), 98.7 (q, J = 2.0, C-5), 76.9 (C-5’), 53.5 (C-7), 37.7 (C-4’);
1
Anal. calcd. for C H F N O (341.08): C, 52.79; H, 3.25; N, 12.31. Found: C, 52.91;
1
5
11
4
3
2
H, 3.17; N, 12.21%.
5
.1.2.10.
1-((3-(2-Toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9c). White powder, 99% yield, m.p. 142–143 ºC (ethanol); H
NMR (400 MHz, DMSO) δ 8.58 (d, J = 6.6, 1H, H-6), 7.43 (d, J = 7.5, 1H, Ar), 7.36 –
7
1
4
1
1
5
.25 (m, 3H, Ar), 6.90 (d, J = 6.6, 1H, H-5), 5.11 – 5.02 (m, 1H, H-5’), 4.28 (dd, J =
3.5, 3.8, 1H, H-7), 4.15 (dd, J = 13.5, 7.6, 1H, H-7), 3.68 (dd, J = 17.2, 10.6, 1H, H-
1
3
’), 3.37 (dd, J = 17.2, 5.8, 1H, H-4’), 2.44 (s, 3H, Me); C NMR (101 MHz, DMSO) δ
61.4 (q, J = 35.4, C-4), 157.7 (C-3’), 155.3 (C-6), 154.5 (C-2), 136.9, 131.2, 129.4,
29.1, 128.0, 125.9 (Ar), 119.4 (q, J = 277.5, CF ), 98.8 (d, J = 1.7, C-5), 75.8 (C-5’),
3
3.5 (C-7), 40.2 (C-4’), 22.0 (Me); Anal. calcd. for C H F N O (337.10): C, 56.97;
1
6
14
3
3
2
H, 4.18; N, 12.46%. Found: C, 56.52; H, 4.22; N, 12.35%.
5
.1.2.11.
1-((3-(4-Toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9d). White powder, 86% yield, m.p. 205–207 ºC (ethanol); H
NMR (400 MHz, DMSO-d /TMS) δ 8.51 (d, J = 6.6, 1H, H-6), 7.53 (d, J = 8.1, 2H,
6
Ar), 7.25 (d, J = 8.0, 2H, Ar), 6.87 (d, J = 6.6, 1H, H-5), 5.10 – 5.02 (m, 1H, H-5’), 4.26
(dd, J = 13.6, 3.7, 1H, H-7), 4.08 (dd, J = 13.6, 8.0, 1H, H-7), 3.58 (dd, J = 17.3, 10.6,
1
3
1
H, H-4’), 3.30 (dd, J = 17.3, 6.1, 1H, H-4’), 2.33 (s, 3H, Me); C NMR (101 MHz,
DMSO-d /TMS) δ 161.6 (q, J = 35.3, C-4), 157.1 (C-3’), 155.3 (C-6), 154.7 (C-2),
6
1
40.3, 129.5, 126.8, 126.2 (Ar), 119.6 (q, J = 277.5, CF ), 99.0 (q, J = 1.8, C-5), 76.8
3
1
3

ACCEPTED MANUSCRIPT
(
C-5’), 53.9 (C-7), 38.0 (C-4’), 21.0 (Me); Anal. calcd. for C H F N O (337.10): C,
1
6
14
3
3
2
5
6.97; H, 4.18; N, 12.46%. Found: C, 57.18; H, 4.03; N, 12.48%
5
.1.2.12. 1-((3-(2-Hydroxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9e). Beige powder, 55% yield, m.p. 212–213 ºC (ethanol); H
NMR (400 MHz, DMSO-d /TMS) δ 9.73 (s, 1H, OH), 8.54 (d, J = 6.6, 1H, H-6), 7.44
6
(dd, J = 7.8, 1.6, 1H, Ar), 7.32 (ddd, J = 8.3, 7.3, 1.7, 1H, Ar), 6.98 – 6.88 (m, 2H, Ar),
6
4
6
.87 (d, J = 6.6, 1H, H-6), 5.13 – 5.03 (m, 1H, H-5’), 4.28 (dd, J = 13.6, 3.8, 1H, H-7),
.14 (dd, J = 13.6, 7.9, 1H, H-7), 3.71 (dd, J = 17.6, 10.5, 1H, H-4’), 3.44 (dd, J = 17.6,
1
3
.1, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 161.3 (q, J = 35.2, C-4), 157.9
6
(Ar), 156.0 (C-3’), 155.0 (C-6), 154.3 (C-2), 131.4, 128.9 (Ar), 119.3 (q, J = 277.5,
CF ) 119.2, 116.2, 114.5 (Ar), 98.6 (d, J = 1.7, C-5), 76.1 (C-5’), 53.3 (C-7), 38.9 (C-
3
4
5
’); Anal. calcd. for C H F N O (339.08): C, 53.10; H, 3.57; N, 12.39%. Found: C,
15 12 3 3 3
2.77; H, 3.76; N, 12.08%
5
.1.2.13. 1-((3-(2-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9f). White powder, 92% yield, m.p. 142–143 ºC (chloroform); H
NMR (400 MHz, DMSO-d /TMS) δ 8.54 (d, J = 6.6, 1H, H-6), 7.50 (d, J = 7.6, 1H,
6
Ar), 7.43 (t, J = 7.9, 1H, Ar), 7.10 (d, J = 8.4, 1H, Ar), 6.97 (t, J = 7.5, 1H, Ar), 6.90 (d,
J = 6.6, 1H, H-5), 5.09 – 5.01 (m, 1H, H-5’), 4.23 (dd, J = 13.5, 3.5, 1H, H-7), 4.13 (dd,
J = 13.6, 7.3, 1H, H-7), 3.82 (s, 3H, OMe), 3.60 (dd, J = 17.8, 10.6, 1H, H-4’), 3.38 –
1
3
3
1
.30 (m, 1H, H-4’); C NMR (101 MHz, DMSO-d /TMS) δ 161.42 (q, J = 35.4, C-4)
6
57.3 (Ar), 156.2 (C-3’), 155.4 (C-6), 154.6 (C-2), 131.7, 129.0, 120.5 (Ar), 119.51 (q,
J = 277.5, CF ), 117.9, 112.2 (Ar), 98.8 (d, J = 1.8, C-5), 76.8 (C-5’), 55.7 (OMe), 53.5
3
1
4

ACCEPTED MANUSCRIPT
(
C-7), 40.1 (C-4’); Anal. calcd. for C H F N O (353.10): C, 54.39; H, 3.99; N,
1
6
14
3
3
3
1
1.89%. Found: C, 54.42; H, 3.89; N, 11.92%.
5
.1.2.14. 1-((3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)
1
pyrimidin-2(1H)-one (9g). White powder, yield 99%, m.p. 188–190 ºC (chloroform). H
NMR (400 MHz, DMSO-d /TMS) δ 8.56 (d, J = 6.6, 1H, H-6), 7.60 (d, J = 8.6, 2H,
6
Ar), 7.01 (d, J = 8.6, 2H, Ar), 6.90 (d, J = 6.6, 1H, H-5), 5.09 – 5.01 (m, 1H, H-5’), 4.26
(dd, J = 13.5, 3.7, 1H, H-7), 4.08 (dd, J = 13.5, 8.1, 1H, H-7), 3.80 (s, 3H, OMe), 3.58
1
3
(
dd, J = 17.2, 10.5, 1H, H-4’), 3.35 – 3.27 (m, 1H, H-4’); C NMR (101 MHz, DMSO-
d6/TMS) δ 161.4 (q, J = 35.4, C-4), 160.8 (Ar), 156.5 (C-3’), 155.3 (C-6), 154.5 (C-2),
1
5
5
28.3, 121.3 (Ar), 119.48 (q, J = 277.3, CF ), 114.2 (Ar), 98.8 (d, J = 1.6, C-5), 76.4 (C-
3
’), 55.3 (OMe), 53.7 (C-7), 37.9 (C-4’); Anal. calcd. for C H F N O (353.10): C,
1
6
14
3
3
3
4.39; H, 3.99; N, 11.89%. Found: C, 54.42; H, 3.86; N, 11.87%.
5
5
.2. Biologic activities
.2.1. General
Dulbecco’s modified Eagle’s medium (DMEM); Roswell Park Memorial Institute
medium (RPMI-1640); phosphate-buffered saline (PBS) — Na HPO , KH PO , and
2
4
2
4
KCl, pH 7.4; and mitoxantrone (MXT) were purchased from Sigma (St. Louis, MO,
USA). Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from
Gibco-BRL (Grand Island, NY, USA). The Cell Proliferation Kit II (XTT) was acquired
from Roche (Basel, Switzerland). All other reagents were of analytical grade.
The human cell lines MCF-7 (breast adenocarcinoma), HepG-2 (hepatoma), T-24
(bladder cancer), CACO-2 (colorectal adenocarcinoma), and HEK-293 (human
embryonic kidney) were obtained from the Rio de Janeiro Cell Bank (Rio de Janeiro,
1
5

ACCEPTED MANUSCRIPT
RJ, Brazil). The HCT-116 cell (colorectal carcinoma) was kindly provided by Dr.
Annette K. Larsen — Institut National de la Santé et de la Recherche Médicale
(
INSERM), Paris, France. All cell lines except MCF-7 were grown in DMEM
−
1
supplemented with 10 % or 20 % (for CACO-2) FBS, 100 units.mL penicillin, and
−
1
1
00 ꢀg.mL streptomycin, at 37 °C in a humidified atmosphere of 5 % CO . MCF-7
2
cells were maintained in RPMI-1640 supplemented with 20 % FBS, in the same
conditions as described above.
5
.2.2. Cytotoxic activity of 1-(3-aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-
1
H-pyrimidin-2-ones 8-9a-g
The cytotoxic potential of the 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones 8-9a-g as evaluated by the XTT assay in human
4
tumor cell lines as well as in HEK-293 normal epithelial cells. Cells (1 × 10 cells) were
seeded on 96-well plates in growth medium and incubated overnight. Afterwards, these
compounds (0.1, 0.5, 1.0, 2.5, 5.0, and 10 ꢀM) were added to each well and incubated
for 24 h. Mitoxantrone, which is a cytostatic anthracenedione that intercalates in DNA
and increases the incidence of double-strand breaks by stabilizing the cleavable
complex of topoisomerase II and DNA, was used as the positive control [23]. At the end
of each treatment, cell viability was assessed according to the manufacturer’s
instructions. Briefly, after discarding the medium, 1 mL of XTT labeling mixture was
added to the cells and incubated for 2 h at 37 °C. Absorbance was measured with a
SpectraMax reader (Bio-Rad, USA) at a test wavelength of 492 nm (A ) and a
4
92
reference wavelength of 690 nm (A ). The final result corresponds to A -A . The
6
90
492
690
absorbance of negative control cells was set as 100 % viability, and the values for
treated cells were calculated as a percentage of the control cells [7].
1
6

ACCEPTED MANUSCRIPT
The IC (50 % inhibitory concentration) values and their 95 % confidence intervals (CI
5
0
9
5 %) were obtained by nonlinear regression, using the GraphPad Prism v5 program
(
Intuitive Software for Science, San Diego, CA, USA). The SI was calculated by IC in
5
0
HEK-293 cells/IC in tumoral cells. All experiments were independently repeated at
5
0
least three times, with triplicate samples for each treatment. Results are expressed as
means ± standard deviation (SD). Data were analyzed by one-way analysis of variance
(ANOVA), and means were compared using the Tukey test, with P ≤ 0.05 considered to
be statistically significant.
Acknowledgement
The authors thank the financial support from the Fundação de Apoio à Tecnologia e
Ciência do Estado do Rio Grande do Sul (FAPERGS, PqG 11/1608-8 and
PRONEX/FAPERGS/CNPq grant No. 10/0044-3), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq – Universal grant No. 476341/2013-
2
and CNPq/INCT/INPeTAm grant No. 573695/2008-3), and Programa Nacional de
Cooperação Acadêmica/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CAPES). The authors also thank fellowships from CAPES (C. M. V), CNPq (M. M. L
(
and J. M. S.) and FAPERGS.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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pyrazolines, Tetrahedron 55 (1999) 345–352.
[
22] a) C.J.A. Ribeiro, J.D. Amaral, C.M.P. Rodrigues, R. Moreira, M.M.M. Santos,
Synthesis and evaluation of spiroisoxazoline oxindoles as anticancer agents,
Bioorg. Med. Chem. 22 (2014) 577584;
b) L. Schwarz, U. Girreser, B. Clement, Synthesis and characterization of para-
substituted N,N’-dihydroxybenzamidines and their derivatives as model
compounds for a new class of prodrugs, Eur. J. Org. Chem. (2014) 1961–1975.
23] F. Hagemeister, F. Cabanillas, M. Coleman, S.A. Gregory, P.L. Zinzani, The role of
mitoxantrone in the treatment of indolent lymphomas, Oncologist 10 (2005) 150-
[
1
59.
2
1

ACCEPTED MANUSCRIPT
Captions:
Table 1. Optimization of the reaction conditions for the synthesis of compound 8a
Table 2. Optimized reaction conditions and yields for the synthesis of compounds 8-9a-g
Table 3. Antiproliferative activity of 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones compounds on human cell lines
Figure 1. Structure of 5-fluorouracil (a), da 6-mercaptopurine (b) e da cytarabine (c)
Figure 2. Structural modifications in the deoxycytidine nucleoside feature
Figure 3. Different types of HOMO-LUMO interactions that control regioselectivity in 1,3-
dipolar cycloaddition reactions [18]
Figure 4. ORTEP of compound 8d
Scheme 1. Convergent synthesis of 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones (8-9a-g)
2
2

ACCEPTED MANUSCRIPT
Table 1. Optimization of the reaction conditions for the synthesis of compound 8a
Entry
Solvent
DCM
DCM
DCM
DCE
Temp.
r.t.
Time (h)
Product (%)
4 + 6a + 8a
4 + 6a + 8a
4 + 6a + 8a
4 + 6a + 8a
8a
Yield (%)
a
1
3
2
4
5
6
20
36
20
20
20
-
a
r.t.
-
a
Reflux
Reflux
r.t.
-
b
-
THF
86
98
THF
Reflux
4
8a
a
1
Proportion of 8a:4 was 1:0.4 determined by H NMR integrals.
b
1
Proportion of 8a:4 was 1:0.2 determined by H NMR integrals.
2
3

ACCEPTED MANUSCRIPT
Table 2. Optimized reaction conditions and yields for the synthesis of compounds 8-9a-g
CX3
CX3
HO
H
N
N
N
i, ii
Y
N
O
O
+
N
O
5
5 - 99%
N
6a-g
Y
8-9a-g
4-5
i = THF, NCS, HCl, r.t., 2 h
ii = Et N, reflux, 4 h.
3
Reactants
Product
Y
X
Yield (%)
4
4
4
4
4
4
4
5
5
5
5
5
5
5
+ 6a
+ 6b
+ 6c
+ 6d
+ 6e
+ 6f
+ 6g
+ 6a
+ 6b
+ 6c
+ 6d
+ 6e
+ 6f
+ 6g
8a
8b
8c
8d
8e
8f
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
98
83
97
80
61
99
94
92
84
99
86
55
92
99
4-F
2-Me
4-Me
2-OH
2-OMe
4-OMe
Ph
8g
9a
9b
9c
9d
9e
9f
4-F
F
2-Me
4-Me
2-OH
2-OMe
4-OMe
F
F
F
F
9g
F
2
4

ACCEPTED MANUSCRIPT
Table 3. Antiproliferative activity of 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-
trihalomethyl-1H-pyrimidin-2-ones compounds on human cell lines
a
IC (μM)
5
0
b
Entry Compd.
Selectivity index (SI)
HEK-293 MCF-7 HepG-2
T-24
HCT-116 CACO-2
1
.04
1
8a
14.63
7.09
5.10
4.15
> 10
(
SI: 14.06)
2
3
8b
8d
5.01
3.59
> 10
> 10
> 10
> 10
> 10
> 10
7.21
> 10
4.95
> 10
1
.37
4
8e
6.12
6.02
6.77
8.50
5.27
(
SI: 4.47)
> 10
5.84
5
6
7
8
9
8f
8g
19.41
5.19
4.30
5.89
> 10
> 10
7.99
> 10
> 10
> 10
0.87
> 10
8.21
> 10
> 10
6.03
5.99
7.03
7.87
3.50
> 10
9.02
> 10
> 10
> 10
6.91
> 10
> 10
8.54
8.96
> 10
> 10
0.61
9a
2.42
> 10
> 10
4.98
9b
5.99
9c
10.85
27.79
10.79
8.43
1.29
SI: 8.4)
> 10
(
1
0
1
2
4
9d
> 10
> 10
> 10
2.40
1
1
1
9f
> 10
> 10
2.50
9g
c
MXT
2.88
a
Drug concentration required to inhibit the cell growth by 50% after 24 h of incubation.
b
Selectivity index (in vitro): IC in HEK-293 cells/IC in tumoral cells. Data represent mean ±
5
0
50
c
three separate experiments. Mitoxantrone (MXT) was used as positive control.
2
5

ACCEPTED MANUSCRIPT
NH2
N
O
SH
N
F
N
NH
N
N
O
OH
N
H
O
N
H
O
HOH C
OH
2
(a)
(b)
(c)
Figure 1. Structure of 5-fluorouracil (a), da 6-mercaptopurine (b) e da cytarabine (c)
2
6

ACCEPTED MANUSCRIPT
Trihalomethyl groups:
-
-
-
Increased stability
Increased lipophilicity
Little steric requirements
NH2
N
N
O
HO
Methylenic Spacer:
Improved resistance to acidic
hydrolysis
-
O
H
H
H
H
H
OH
Heterocyle Mod if ication: dihydroisoxazolidine
-
-
-
Less basic nitrogen atom
Lower nucleophilicity of oxygen atom
Increased stability
Figure 2. Structural modifications in the deoxycytidine nucleoside feature
2
7

ACCEPTED MANUSCRIPT
Het
O
N
C
Het
O
N
Ar
Ar
Dipolarophile Dipole
3
,5-disubstituted isoxazolines
(HOMO)
(LUMO)
O
N
C
Het
Ar
N
Het
Ar
O
Dipolarophile
LUMO)
Dipole
3
,4-disubstituted isoxazolines
(
(HOMO)
Figure 3. Different types of HOMO-LUMO interactions that control regioselectivity in
,3-dipolar cycloaddition reactions [18]
1
2
8