Synthesis and biological evaluation of 1,7,8,8a-tetrahydro-3H-oxazolo[3,4- a]pyrazin-6(5H)-ones as antitumoral agents

Article abstract of DOI:10.1016/j.bmc.2013.07.019

A series of 1,7,8,8a-tetrahydro-3H-oxazolo[3,4-a]pyrazin-6(5H)-ones has been synthesized by an intramolecular, palladium(II) catalyzed, aminooxygenation of alkenyl ureas, readily available from glycine allylamides as starting materials. Biological tests s

Full text of DOI:10.1016/j.bmc.2013.07.019

Bioorganic & Medicinal Chemistry 21 (2013) 5748–5753
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Bioorganic & Medicinal Chemistry
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Synthesis and biological evaluation of 1,7,8,8a-tetrahydro-
3H-oxazolo[3,4-a]pyrazin-6(5H)-ones as antitumoral agents
a
a
b
a
Ugo Chiacchio a, , Vincenzina Barbera , Roberta Bonfanti , Gian Luigi Broggini , Agata Campisi ,
⇑
Silvia Gazzola b, Rosalba Parenti c, Giovanni Romeo d,
⇑
a Dipartimento di Scienze del Farmaco, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy
b Dipartimento di Scienza e Alta Tecnologia, Università dell’Insubria, Via Valleggio 11, 22100 Como, Italy
c Dipartimento di Scienze Bio-Mediche, Università di Catania, Sezione di Fisiologia, Via A. Doria 6, 95125 Catania, Italy
d Dipartimento di Scienze del Farmaco e Prodotti Per la Salute, Università di Messina, Via SS Annunziata, 98168 Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,7,8,8a-tetrahydro-3H-oxazolo[3,4-a]pyrazin-6(5H)-ones has been synthesized by an intra-
molecular, palladium(II) catalyzed, aminooxygenation of alkenyl ureas, readily available from glycine
allylamides as starting materials. Biological tests showed that the obtained compounds are endowed with
an interesting antitumoral activity against two human thyroid cancer cell lines, namely FTC-133 and
8305C, by promoting the apoptotic pathway and DNA fragmentation.
Received 14 May 2013
Revised 8 July 2013
Accepted 9 July 2013
Available online 18 July 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Palladium
Domino reaction
Bicyclic piperazinones
Thyroid cancers
Caspase-3
1. Introduction
The piperazine ring is also used as both rigid-dimensional dia-
mine core structure, as well as a modifying group to introduce an
Cancer has become one of the leading disease-related causes of
death of human population.1 Among all the current therapeutic
methods, chemotherapy remains an important option for cancer
treatment: inhibition of the characteristic proliferation pathways
of cancer cells constitutes an effective strategy to fight this pathol-
ogy.2 Great research has been devoted to the discovery of new and
selective anticancer agents; however, the development of drug and
multidrug resistance, in which cancer cells become resistant to dif-
ferent structural types of chemotherapeutic agents, is one of the
major hindrances to successful cancer chemotherapy.3 Thus, the
discovery and identification of novel compounds with different
characteristic than the currently used or showing an alternative
mechanism of action, is an intensively explored research area in
medicinal chemistry.4
amino residue in the molecule.6 The incorporation of a further het-
erocycle ring into the prospective pharmaceutical candidate (i.e.
the piperazine derivative) is a well-known tactic to gain activity
and safety advantages. In this context, much work has been direc-
ted towards the design and the synthesis of novel fused-piperazine
derivatives.7
As part of our medicinal chemistry program aimed at the discov-
ery of potential anticancer agents,8 we have addressed our attention
to the synthesis of a novel series of derivatives containing the oxaz-
olo–pyrazine pharmacophore.9 The oxazole unit constitutes the
core structure of several biological active compounds: oxazole con-
taining heterocycle compounds play an important role in medicinal
chemistry as antimicrobial, CNS agents, antihyperglycemic
Nitrogen heterocycles are found in the majority of clinically ap-
proved drugs.5 One of important pharmacologically relevant het-
erocycles is the piperazine ring system, which is found in ca. 7%
of all small therapeutic molecules approved by the FDA, starting
from the year 2000.
⇑
Corresponding authors. Tel.: +39 095 7385013; fax: +39 095 580138 (U.C.).
E-mail address: uchiacchio@unict.it (U. Chiacchio).
Figure 1. Structures of some anticancer agents containing the oxazolo–piperazine
core.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.019

U. Chiacchio et al. / Bioorg. Med. Chem. 21 (2013) 5748–5753
5749
potentiating compounds, antitumor, analgesics and anti-inflamma-
tory agents.10 Quinocarcin,11 which has shown remarkable antipro-
liferative activity against lymphocytic leukemia, and tetrazomine, a
potent antitumoral antibiotic,12 (Fig. 1) are two relevant examples
of compounds containing an oxazolo–piperazine core.
strictly close to those of analogous 3H-oxazolo[3,4-a]pyrazine
derivatives, previously obtained by cyclization of Boc-glycine ally-
lamides.18 On the other hand, in the 13C NMR spectra, the reso-
nance of the methylene group of the five-membered ring, in the
range 67.7–68.0 ppm, is consistent with the 4,5-dihydro-oxazole
ring. These values allow to exclude an imidazolidinone ring,
whose methylene group is reported to resonate in the range of
40–50 ppm.21
On the other hand, small molecules targeting DNA/RNA have at-
tracted significant scientific interest due to the medicinal, bio-
chemical and biological implications of such molecular
recognition events. The design and the synthesis of novel molecu-
lar entities, with multiple DNA/RNA binding features and with a
structurally well-defined architecture, is still highly demanding.13
On the basis of these consideration, we report here the synthe-
sis and the biological evaluation of oxazolo–piperazine hybrid
compounds in which an oxazolo–pyrazine unit is linked to an aryli-
mine moiety. The synthetic approach exploits the intramolecular,
palladium(II) catalyzed, aminooxidation of alkenyl ureas, prepared
starting from glycine allylamides. The biological effects of the new
obtained compounds have been evaluated on human follicular
(FTC-133) and anaplastic (8305C) thyroid carcinoma cell lines, as
representatives of two aggressive types of thyroid cancer, poorly
differentiated and dedifferentiated, respectively.14–16
The aminooxygenation products 11a–c and 12 are explainable
by the attach of the oxygen atom on the
r-alkylcomplex 13, gen-
erated by aminopalladation of the amides 9 and 10 (Scheme 2).
In principle, according to the nucleophilic behavior of secondary
amides in transition metal-catalyzed reactions,22 both oxygen
and nitrogen atoms of the urea group could be involved in the sec-
ond intramolecular step, leading to the formation of an oxazole or
an imidazole ring fused to the piperazinone moiety.
However, our experimental conditions have allowed a complete
control of two alternative reaction pathways, with the exclusive for-
mation of the target oxazolo–piperazine conjugates 11a–c and 12.
2.2. Biological evaluation
2.2.1. Cytotoxic effect of the compounds
2. Results and discussion
2.1. Chemistry
To evaluate the antitumor potential of compounds 11a–c and
12, we used two cancer cell lines, FTC-133 and 8305C, as represen-
tative of follicular and anaplastic thyroid human cancer, respec-
tively. To monitor cell viability, both fluorescent microscope
analysis and MTT assays have been used. In preliminary experi-
ments, FTC-133 and 8305C cell line cultures were exposed to dif-
The preparation of the 1,7,8,8a-tetrahydro-3H-oxazolo[3,4-
a]pyrazines 11a–c and 12 is shown in Scheme 1: the key step of
the synthetic process exploits a transition metal-catalyzed double
intramolecular domino process.17 Thus, the N-allyl-amides of Boc-
glycine 4 and 5, obtained by the reaction of N-Boc-glycine 1 with
cyclohexyl- or phenyl-allylamine 2 and 3, respectively, in the pres-
ence of DCC and DMAP,18 were converted to the corresponding al-
lyl amides 6 and 7, by removal of the t-butoxycarbonyl group, and,
then, treated with the appropriate commercially available or easily
prepared aryl isocyanates 8a–c,19 respectively.
The cyclization of the alkenyl ureas 9a–c and 10 was conve-
niently performed working with a PdCl2(MeCN)2/CuCl2 catalytic
system, already reported in literature as an useful tool for domino
reactions of alkenyl derivatives with nucleophiles.20 Indeed, bicy-
clic ring-fused piperazinones 11a–c and 12 were achieved in high
yield with PdCl2(MeCN)2 as the catalyst (5 mol %) and CuCl2
(3 equiv) in THF under reflux at 60 °C for 12 h.
ferent concentrations (5–100 lM) of the synthesized compounds
11a–c and 12 for 24 h, in order to establish the optimal concentra-
tion.23–25 A significant reduction in cellular viability was observed
in both FTC-133 and 8305C cell lines treated with the compounds
11a–c and 12, at 50 lM concentrations for 24 h, when compared
with the respective untreated cell cultures used as controls. The
50% cytotoxic inhibitory concentration (IC50), causing 50% decreas-
ing in cell proliferations, obtained graphically from dose-effect
curves using Prism 5.0 (GraphPad Software Inc.), is reported in Ta-
ble 1. All compounds displayed cytotoxic effects on the both cell
lines, at concentrations ranging from 44 to 80 lM. FTC-133 cells
were more susceptible to treatment with compounds 11a–c and
12, than the 8305C cells.
2.2.2. Evaluation of the apoptotic pathway activation
To investigate whether apoptosis was triggered by
treatment with the new compounds, caspase-3 cleavage by
The structure of the obtained compounds was assigned on the
basis of spectroscopic data. In particular, in the 1H NMR spectra,
chemical shifts attributable to the resonance of all protons are
Scheme 1. Synthesis of 1,7,8,8a-tetrahydro-3H-oxazolo[3,4-a]pyrazine compounds 11a–c and 12.

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U. Chiacchio et al. / Bioorg. Med. Chem. 21 (2013) 5748–5753
Scheme 2. Selective formation of aminooxygenation products 11a–c and 12.
Table 1
Concentrations (
l
M) of investigated compounds 11a–c, and 12 that induced 50% decrease (IC50) in FTC-133 and 8305C cell proliferationa
Cell lines
Untreated (control)
11a
11b
11c
12
FTC–133
8305C
>100
>100
50.03 3.90
70.66 2.13
44.79 4.27
61.03 5.27
48.32 2.98
68.45 3.71
74.21 3.93
80.28 4.52
a
FTC–133 and 8305C cell lines were incubated with drug compounds in concentration ranging from 5 to 100 lM at 37 °C in a 5% CO2 atmosphere for 24 h. Viability was
determined by the MTT assay. Each data represents mean SD from four independent experiments, performed in triplicate.
immunocytochemical analysis was evaluated after 24 h of cell
Table 2
incubation at 50 lM concentration of compounds 11a–c and 12.
Quantification and statistical analysis of caspase-3 positive cells in FCT–133 and
8305C human thyroid cancer cell line cultures untreated (Control) and treated with
Significant enhancement of caspase-3 positive cells was detected
after 24 h of treatment in FTC-133 and 8305C cell lines, when com-
pared to the untreated control. The effect appeared more evident in
FTC-133 cell lines, with compound 12 showing the lowest activity
with respect 11a–c (Fig. 2A).
50
l
M 11a–c or 12 for 24 h.
% Caspase-3 positive FTC–133
% Caspase-3 positive 8305C
cell lines
cell lines
Treatment 24 h
24 h
Control
11a
11b
11c
2.51
1
88.68 1*
90.56 2*
89.24 2*
29.30 3*
3.22 1*
75.88 1*
78.56 2*
68.30 3*
18.24 2*
The quantification and statistical analysis of caspase-3 immu-
nolabeling is reported in Table 2.
2.2.3. Evaluation of DNA fragmentation and molecule/DNA
labeling
12
*
p < 0.05 versus respective control by one-way ANOVA followed by post hoc
We tested DNA fragmentation and the eventual molecule/DNA
labeling in untreated and treated FTC-133 and 8305C thyroid can-
cer cell lines. We observed that all the molecules tested, and prev-
alently 11a–c compounds, were able to induce DNA fragmentation
in treated cancer cell lines. The effect appeared more evident in
FTC-133 cancer cell lines. Furthermore, to assess the molecule/
DNA labeling, gel electrophoresis was performed in absence or in
presence of SYBR Green I, a well-known fluorescent dye that binds
specifically to double-stranded DNA (Fig. 3). Molecule/DNA label-
ing was detected only in presence of the fluorescent dye. The ob-
tained data show that all the molecules induced DNA
fragmentation at a different degree.
Holm–Sidak test. Data were collected from 4 fields/coverslip of four experiments in
duplicate.
According to this test, our data suggest a weak stacking interac-
tion between the compounds 11a–c, 12, and the DNA bases, prob-
ably due to a hydrogen bond between the amido group of the
piperazine group and the amino group of poly-d(AT) and/or poly-
d(GC) chain lying in the minor groove. Furthermore, the low activ-
ity observed in both cancer cell line cultures for compound 12,
which possesses a phenyl ring into the amido portion of the mole-
cule, might be related to a reduced electron releasing effect (cross
Figure 2. (A) Fluorescent microscope analysis of caspase-3 cleavage in (A) FCT-133 and (B) 8305C human thyroid cancer cell lines, untreated (control) and treated with 50
11a–c or 12 for 24 h. Scale bars = 50 m.
lM
l

U. Chiacchio et al. / Bioorg. Med. Chem. 21 (2013) 5748–5753
5751
7.64 (br s, 1H); minor rotamer: d 1.07–1.82 (m, 10H), 3.47–3.59
(m, 1H), 3.94 (d, J = 5.1 Hz, 2H), 4.23 (s, 2H), 5.06–5.26 (m, 2H),
5.72–5.87 (m, 1H), 6.45 (br s, 1H), 7.00–7.03 (m, 1H), 7.24–7.28
(m, 2H), 7.32–7.36 (m, 2H), 7.64 (br s, 1H); 13C NMR (125 MHz,
CDCl3) major rotamer: d 25.3, 25.5, 29.7, 44.7, 46.1, 52.4, 117.5,
118.5, 121.1, 128.8, 129.1, 137.7, 155.5, 164.4; minor rotamer: d
25.3, 25.4, 29.3, 44.5, 46.1, 47.6, 116.8, 118.5, 121.1, 128.8, 129.1,
138.3, 155.6, 164.4. MS: m/z 315 (M+). Anal. Calcd for
C18H25N3O2: C, 68.54; H, 7.99; N, 13.32. Found C, 68.72; H, 7.73;
N, 13.41.
Figure 3. DNA fragmentation in untreated or treated FTC-133 (left) and 8305C
(right) cells with 50 of 11a–c and 12 for 24 h. Gel electrophoresis was
lM
performed in presence of SIBER Green I. Glyceraldehyde-3-phosphate dehydroge-
nase-1 (GAPDH-1) gene was used as a reference gene to normalize target gene
expression.
4.1.2. N-Allyl-N-cyclohexyl-2-(3-naphthalen-1-ylureido)-
acetamide (9b)
Eluent: light petroleum–AcOEt 3:2. Beige solid; yield 75%, mp
173–174 °C. IR (nujol):
m ;
= 3287, 1745, 1656 cmÀ1 1H NMR
conjugation) which decreases the ability to form a hydrogen bond
with the DNA-nucleobases.
(500 MHz, CDCl3, mixture of two rotamers in 4:3 ratio) major rot-
amer: d 1.02–1.79 (m, 10H), 3.74 (d, J = 4.8 Hz, 2H), 4.11 (s, 2H),
4.19–4.32 (m, 1H), 4.98–5.16 (m, 2H), 5.56–5.72 (m, 1H), 6.73 (br
s, 1H), 7.42–7.50 (m, 3H), 7.63 (br s, 1H), 7.82–7.86 (m, 2H),
8.08–8.13 (m, 2H); minor rotamer: d 1.02–1.79 (m, 10H), 3.39–
3.45 (m, 1H), 3.81 (d, J = 5.4 Hz, 2H), 4.20 (s, 2H), 4.98–5.16 (m,
2H), 5.56–5.72 (m, 1H), 6.81 (br s, 1H), 7.42–7.50 (m, 3H,), 7.67
(br s, 1H), 7.82–7.86 (m, 2H), 8.08–8.13 (m, 2H); 13C NMR
(125 MHz, CDCl3) major rotamer: d 25.3, 25.5, 29.6, 42.6, 44.8,
54.3, 114.1, 116.9, 121.3, 122.0, 125.2, 125.8, 126.0, 128.3, 128.5,
134.1, 134.3, 157.0, 169.6; minor rotamer: d 25.3, 25.4, 29.2,
42.4, 44.3, 56.4, 114.1, 116.1, 121.1, 121.9, 125.1, 125.5, 125.9,
128.3, 128.5, 133.7, 134.5, 156.9, 168.4. MS: m/z 365 (M+). Anal.
Calcd for C22H27N3O2: C, 72.30; H, 7.45; N, 11.50. Found C, 72.09;
H, 7.61; N, 11.73.
3. Conclusions
In summary, we report an efficient synthesis of a series of oxaz-
olo[3,4-a]pyrazin-6(3H)-ones, according to a procedure based on a
double intramolecular domino process catalyzed by PdCl2(MeCN)2/
CuCl2 performed on different alkenyl ureas. Biological tests indi-
cate that the obtained compounds are endowed with an interesting
antitumor activity against two thyroid carcinoma cell lines, by acti-
vating caspase-3 dependent apoptotic pathway and DNA fragmen-
tation with a prevalent action on follicular type.
4. Experimental section
4.1.3. N-Allyl-N-cyclohexyl-2-(3-pyren-1-ylureido)-acetamide
(9c)
Flash column chromatography was performed employing 230–
400 mesh silica gel. Analytical thin layer chromatography was per-
formed on silica gel 60 F254. Melting points were measured with a
Büchi B-540 apparatus and are uncorrected. IR spectra were re-
corded on a Nicolet 550 FT-IR spectrophotometer. Nuclear mag-
netic resonance spectra were acquired on Varian instrument at
200 or 500 MHz for 1H NMR and 125 MHz for 13C NMR. 13C spectra
were 1H decoupled and multiplicities were determined by the APT
pulse sequence. EI mass spectra were recorded at an ionizing volt-
age of 6 KeV on a VG 70-70 EQ. Elemental analyses were executed
on a Perkin–Elmer CHN Analyzer Series II 2400.
Eluent: light petroleum–AcOEt 3:2. Brown solid; yield 70%, mp
97–99 °C. 1H NMR (200 MHz, CDCl3): d 1.02–1.79 (m, 10H), 3.58–
3.69 (m, 1H), 3.94–4.02 (m, 1H), 4.44 (d, 1H), 4.53 (d, 1H), 5.10–
5.37 (m, 2H), 5.72–5.90 (m, 2H), 7.34 (br d, 1H), 8.00–8.24 (m,
9H), 8.70 (d, 1H); 13C NMR (125 MHz, CDCl3): d 24.9, 25.8, 29.2,
29.7, 43.1, 46.0, 52.5, 118.4, 121.4, 122.8, 124.5, 124.8, 125.0,
125.3, 125.9, 126.5, 127.3, 127.5, 128.0, 129.0, 129.7, 131.0,
131.3, 131.7, 132.1, 140.1, 157.0, 169.3. MS: m/z 439 (M+). Anal.
Calcd for C28H29N3O2: C, 76.51; H, 6.65; N, 9.56. Found C, 76.39;
H, 6.88; N, 9.59.
4.1. General procedure for the preparation of alkenyl ureas 9a–c
and 10
4.1.4. N-Allyl-N-phenyl-2-(3-pyren-1-ylureido)-acetamide (10)
Eluent: light petroleum–AcOEt 3:2. Brown solid; yield 67%, mp
103–105 °C. 1H NMR (200 MHz, CDCl3): d 3.81 (d, J = 4.4 Hz, 1H),
4.15 (d, J = 6.3 Hz, 2H), 4.90–4.99 (m, 2H), 5.61–5.74 (m, 1H),
6.30 (br s, 1H), 7.01–7.05 (m, 2H), 7.23–7.27 (m, 4H), 7.81 (br s,
1H), 7.98–8.05 (m, 3H), 8.11–8.30 (m, 6H); 13C NMR (125 MHz,
CDCl3): d 24.9, 25.8, 29.2, 43.1, 45.8, 52.5, 118.4, 121.2, 122.8,
124.7, 124.7, 125.0, 125.3, 126.0, 126.5, 127.2, 127.5, 128.0,
128.6, 129.8, 130.9, 131.3, 132.1, 140.2, 156.9, 169.2. MS: m/z
433 (M+). Anal. Calcd for C28H23N3O2: C, 77.58; H, 5.35; N, 9.69.
Found C, 77.76; H, 6.11; N, 9.92.
Trifluoroacetic acid (3 mL) was added to a solution of allyl
amide 4 or 5 (1 mmol) in CH2Cl2 (3 mL). The mixture was stirred
at room temperature for 1 h. The solvent was evaporated to dry-
ness and the residue was taken with a 1 M solution of NaOH up
to pH 13. The mixture was extracted with CH2Cl2 (3 Â 10 mL)
and the organic layer was dried over Na2SO4. Concentration at re-
duced pressure gave a colourless oil, which was dissolved in THF
(5 mL) and treated with the appropriate isocyanate 8a–c (1 mmol).
The solution was stirred at room temperature overnight and then
the solvent was evaporated to give a crude residue that was chro-
matographed on silica gel column.
4.2. General procedure for synthesis of 3-arylimino-1,7,8,8a-
tetrahydro-3H-oxazolo[3,4-a]pyrazin-6(5H)-ones 11a–c and 12
4.1.1. N-Allyl-N-cyclohexyl-2-(3-phenylureido)-acetamide (9a)
Eluent: light petroleum–AcOEt 3:2. White solid; yield 82%, mp
97–99 °C. IR (nujol):
(500 MHz, CDCl3, mixture of two rotamers in 7:6 ratio) major rot-
amer: d 1.07–1.82 (m, 10H), 3.91 (d, J = 4.8 Hz, 2H), 4.13 (s, 2H),
4.34–4.46 (m, 1H), 5.06–5.26 (m, 2H), 5.72–5.87 (m, 1H), 6.45 (br
s, 1H), 7.00–7.03 (m, 1H), 7.24–7.28 (m, 2H), 7.32–7.36 (m, 2H)
A solution of the appropriate alkenyl urea 9a–c or 10 (1 mmol)
in dry THF (3 mL) was added to a suspension of PdCl2(MeCN)2
(13 mg, 0.05 mmol) and CuCl2 (402 mg, 3.0 mmol) in dry THF
(3 mL). The reaction was heated at 60 °C for 12 h. After addition
of brine, the mixture was extracted with CH2Cl2 (3 Â 10 mL). The
organic layer was dried over Na2SO4 and the solvent was
m ;
= 3301, 1750, 1654 cmÀ1 1H NMR

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U. Chiacchio et al. / Bioorg. Med. Chem. 21 (2013) 5748–5753
evaporated under reduced pressure to give a crude product that
was purified by silica gel column chromatography.
Foetal Bovine Serum (FBS, GIBCO), Normal Goat Serum (NGS, GIB-
CO), Streptomycin and penicillin antibiotics, Trypsin–EDTA 0.05%
solution were from Invitrogen (Milan, Italy). Lab-Tek™ Chamber
Slides II, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide salts (MTT), agarose, and other chemicals of analytical
grade were obtained from Sigma–Aldrich (Milano, Italy). Mouse
monoclonal antibody against caspase-3 was from Becton–Dickin-
son (Milan, Italy). Tetrarhodamine isothiocyanate (TRITC)-conju-
gated anti-mouse IgG polyclonal antibody, were from Chemicon
(Prodotti Gianni, Milan, Italy). QIAamp DNA Mini kit was from
Qiagen.
4.2.1. 7-Cyclohexyl-3-phenylimino-1,7,8,8a-tetrahydro-3H-
oxazolo[3,4-a]pyrazin-6(5H)-ones (11a)
Eluent: light petroleum–AcOEt 3:7. White solid; yield 85%, mp
155–156 °C. IR (nujol):
m ;
= 1757, 1585 cmÀ1 1H NMR (500 MHz,
CDCl3): d 1.25–1.82 (m, 10H), 3.15 (dd, J = 11.1, 9.3 Hz, 1H), 3.28
(dd, J = 12.0, 3.8 Hz, 1H), 3.82–3.87 (m, 1H), 3.88 (d, J = 17.8 Hz,
1H), 4.02 (dd, J = 8.6, 4.4 Hz, 1H), 4.43 (dd, J = 8.6, 7.4 Hz, 1H),
4.46–4.52 (m, 1H), 4.53 (d, J = 17.8 Hz, 1H) 6.89–6.98 (m, 1H),
7.05 (d, J = 7.6 Hz, 2H), 7.20–7.24 (m, 2H); 13C NMR (125 MHz,
CDCl3): d 25.3, 25.4, 25.6, 29.3, 29.6, 43.6, 46.4, 51.6, 52.6, 67.9,
122.5, 123.4, 128.5, 146.6, 150.9, 164.3. MS: m/z 313 (M+). Anal.
Calcd for C18H23N3O2: C, 68.98; H, 7.40; N, 13.41. Found C, 69.09;
H, 7.17; N, 13.66.
4.3.2. Cell cultures
FTC-133 and 8305C cell lines were suspended in appropriate
medium and plated in flasks at a final density of 2 Â 106 cells or
in Lab-Tek™ Chamber Slides II at a final density 0.5 Â 105 cells/
well. Specifically the medium for FTC-133 cell lines was: DMEM
4.2.2. 7-Cyclohexyl-3-(naphthalen-1-ylimino)-1,7,8,8a-
tetrahydro-3H-oxazolo[3,4-a]pyrazin-6(5H)-ones (11b)
Eluent: light petroleum–AcOEt 3:7. White solid; yield 69%, mp
containing 2 mM Gluta-MAX, 10% FBS, streptomycin (50 lg/mL),
penicillin (50 U/mL); whereas the medium for 8305C cell lines
was: MEM containing 2 mM Gluta-MAX, 10% FBS, streptomycin
(50 lg/mL), penicillin (50 U/mL), and 1% nonessential amino acids.
175–176 °C. IR (nujol):
m
= 1753, 1581 cmÀ1
;
1H NMR (500 MHz,
CDCl3): d 1.05–1.82 (m, 10H), 3.24 (dd, J = 11.3, 10.6 Hz, 1H), 3.34
(dd, J = 12.0, 3.9 Hz, 1H), 3.92–3.98 (m, 1H), 4.02-4.07 (m, 2H),
4.47 (dd, J = 8.7, 7.2 Hz, 1H), 4.53–4.60 (m, 1H), 4.75 (d,
J = 18.3 Hz, 1H), 7.19–7.21 (m, 1H), 7.36–7.45 (m, 3H), 7.52 (d,
J = 8.2 Hz, 1H), 7.29–7.32 (m, 1H), 8.19–8.22 (m, 1H); 13C NMR
(125 MHz, CDCl3): d 25.3, 25.4, 25.6, 29.3, 29.7, 43.6, 46.6, 51.7,
52.6, 67.8, 117.4, 122.6, 124.1, 124.9, 125.7, 125.8, 127.7, 129.3,
134.3, 142.8, 150.9, 164.4. MS: m/z 363 (M+). Anal. Calcd for
Cell lines were then incubated at 37 °C in humidified atmosphere
containing 5% CO2 and the medium was replaced every 2 or 3 days.
When the cultures were about 85–90% confluent, cells were tryp-
sinized by 0.05% trypsin and 0.53 mM EDTA at 37 °C in humidified
atmosphere containing 5% CO2 for 5 min. Trypsinization was
stopped by adding 20% FBS, resuspended and plated in flasks fed
with fresh basic complete media. Cells were seeded again at 1:4
density ratio and incubated at 37 °C in humidified atmosphere con-
taining 5% CO2.
C22H25N3O2: C, 72.70; H, 6.93; N, 11.56. Found C, 72.63; H, 7.19;
N, 11.80.
4.3.3. Treatment of the cells
4.2.3. 7-Cyclohexyl-3-(pyrenyl-1-ylimino)-1,7,8,8a-tetrahydro-
3H-oxazolo[3,4-a]pyrazin-6(5H)-ones (11c)
FTC-133 and 8305C were replated on to Lab-Tek™ Chamber
Slides II at a final density of 1 Â 104 cells/well, and fed in fresh
complete medium. In preliminary experiments, we exposed the
both cultures in the absence or the presence of different concentra-
Eluent: light petroleum–AcOEt 3:7. Yellow bright solid; yield
65%, mp 132–134 °C. 1H NMR (500 MHz, CDCl3): d 1.05–1.82 (m,
10H), 3.34 (dd, J = 11.3, 10.6 Hz, 1H), 3.40 (dd, J = 12.0, 3.9 Hz,
1H), 4.02–4.07 (m, 1H), 4.16–4.12 (m, 2H), 4.56–4.60 (m, 2H),
4.85 (d, J = 18.3 Hz, 1H), 7.19–7.21 (m, 1H), 7.36–7.45 (m, 3H),
7.52 (d, J = 8.2 Hz, 1H), 7.29–7.32 (m, 1H), 8.19–8.22 (m, 2H),
8.45 (d, 1H); 13C NMR (125 MHz, CDCl3): d 25.3, 25.4, 25.6, 29.3,
29.7, 43.7, 46.7, 51.9, 52.6, 68.0, 120.4, 123.6, 124.1, 124.2, 125.0,
125.4, 125.5, 125.7, 126.2, 127.0, 127.4, 131.6, 142.1, 151.8,
164.4. MS: m/z 437 (M+). Anal. Calcd for C28H27N3O2: C, 76.86; H,
6.22; N, 9.60. Found C, 77.05; H, 6.07; N, 9.87.
tions of 11a–c or 12 (5, 10, 25, 50, 75, 100 lM) for 12, 24 h, in order
to establish the optimal concentrations and their exposure times to
all synthesized compounds. For this purpose, MTT test and mor-
phological characterization were utilized.26 We found that for the
both cultures the optimal concentration of all synthesized com-
pounds was 50 lM and the optimal exposure time was 24 h.
4.3.4. MTT bioassay
Cell survival analysis was performed by MTT reduction assay,
evaluating mitochondrial dehydrogenase activity.23,24 Cells were
set up 6 Â 105 cells per well of a 96-multiwell, flat-bottomed,
4.2.4. 7-Phenyl-3-(pyrenyl-1-ylimino)-1,7,8,8a-tetrahydro-3H-
oxazolo[3,4-a]pyrazin-6(5H)-ones (12)
200 lL microplate, and maintained at 37 °C in a humidified 5%
Eluent: light petroleum–AcOEt 3:7. Light yellow solid; yield
67%, mp 93–96 °C. 1H NMR (500 MHz, CDCl3): d 3.58–3.66 (m,
2H), 3.89–3.94 (m, 1H), 4.06–4.09 (m, 2H), 4.52–4.55 (m, 2H),
5.04 (d, J = 18.8 Hz, 1H), 7.32 (d, 2H) 7.44 (t, 2H) 7.86 (d,
J = 7.9 Hz 1H), 7.90–8.01 (m, 4H), 8.1–8.06 (m, 3H), 8.49 (d,
J = 9.2 Hz, 1H); 13C NMR (125 MHz, CDCl3): d 29.7, 31.6, 46.7,
46.9, 51.7, 52.8, 67.7, 96.1, 120.3, 123.6, 124.1, 124.3, 124.7,
125.0, 125.1, 125.5, 125.7, 125.8, 126.2, 126.3, 127.0, 127.4,
127.5, 127.6, 129.4, 129.5, 131.5, 131.6, 140.9, 141.9, 150.8,
165.0. MS: m/z 431 (M+). Anal. Calcd for C28H21N3O2: C, 77.94; H,
4.91; N, 9.74. Found C, 78.01; H, 4.76; N, 9.48.
CO2/95% air mixture.26 At the end of treatment time, 20
lL of
0.5% MTT in (pH 7.4) PBS were added to each microwell. After
1 h of incubation with the reagent, the supernatant was removed
and replaced with 200 lL of dimethyl sulfoxide (DMSO). The opti-
cal density of each well was measured with a microplate spectro-
photometer reader (Titertek Multiskan; Flow Laboratories,
Helsinki, Finland) at 570 nm.
4.3.5. Immunocytochemistry
Expression of caspase-3 in FTC-133 and 8305C cells was identi-
fied by immunocytochemical procedures.24,25 Untreated or 11a–c
and 12 treated FTC-133 and 8305C were fixed by exposing to 4%
paraformaldehyde in 0.1 M PBS for 20 min. Then, cells were
washed three times with PBS and incubated for 1 h at 37 °C in
humidified air and 5% CO2 with 1% NGS in PBS to block unspecific
sites. The cells were successively incubated overnight at 37 °C in
humidified air and 5% CO2 with mouse monoclonal antibody
against caspase-3 (1:200). Finally, the slides were washed three
4.3. Biological assay
4.3.1. Materials
Dulbecco’s modified Eagle medium (DMEM) and Minimum
Essential Medium (MEM) containing 2 mM GlutaMAX (GIBCO),
Ham’s F12 (GIBCO), nonessential amino acids, heat inactivated-

U. Chiacchio et al. / Bioorg. Med. Chem. 21 (2013) 5748–5753
5753
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times with PBS, mounted in PBS/glycerol (50:50), and analyzed on
a Leica fluorescent microscopy (Germany). No nonspecific staining
of hMSCs was observed in control incubations in which the pri-
mary antibody was omitted.
9. Ho, P. S.; Yoon, D. O.; Han, S. Y.; Lee, W. I.; Kim, J. S.; Park, W. S.; Ahn, S. O.; Kim,
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4.3.6. DNA labelling assay
DNA extraction from both untreated and treated FTC-133 and
8305C cells with 75 lM of 11a–c or 12 for 24 h was performed
according to the user’s manual. Gel electrophoresis separates
DNA fragments by size in an 2% agarose gel. DNA is visualized by
including in the gel an intercalating dye, SYBER Green I. GAPDH-
1 as housekeeping gene was used.
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Data were statistically analysed using one-way analysis of var-
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estimate significant differences among groups. Data were reported
as mean SD of four experiments in duplicate, and differences be-
tween groups were considered to be significant at p < 0.05.
Acknowledgments
We gratefully acknowledge the MIUR, the Universities of Cata-
nia, Messina and Insubria and CINMPIS for partial financial
support.
´
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