Synthesis, characterization, crystal structure and cytotoxicity of 2,4-bis(selenomethyl)quinazoline

Article abstract of DOI:10.1007/s11224-011-9816-x

Organoselenium compounds have already been reported to be good anticarcinogenic candidates. A new selenoquinazoline derivative, 2,4-bis(selenomethyl)quinazoline (compound 1), has been synthesized, spectroscopically characterized and its crystal structure has been studied. An intermolecular coupling between C₂ and H′ 5 in the Heteronuclear Multiple Bond Correlation (HMBC) experiment has been observed. Assuming that the headto- tail overlap of parallel molecules (as identified by X-ray diffraction) remains in solution to give bimolecular entities, the p-p interaction enables heteronuclear coupling between the former atoms with a three-bond distance [C₂...(φ-φ)..C′ ₅-H′ ₅]. The crystal structure of compound 1 has been solved by X-ray diffraction. It crystallizes in triclinic system, space group P-1. Unit cell parameters are a = 7.4969(7) A , b = 8.7008(8) A , c = 10.1666(9) A , α = 110.215(2)-, β = 90.354(2)-, c = 115.017(1)-. Linear chains in crystals of compound 1 are generated by C-H...Se and Se..Se bonds between molecules. Furthermore, head-to-tail overlap of parallel molecules, in which p-p interactions can occur, is observed. Compound 1 exhibited a cytotoxic effect in all of the evaluated tumoral cell lines and showed a higher cytotoxic effect in colon and breast cancer cell lines than etoposide, which was used as a reference compound.

Full text of DOI:10.1007/s11224-011-9816-x

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Struct Chem (2011) 22:1233–1240
DOI 10.1007/s11224-011-9816-x
ORIGINAL RESEARCH
Synthesis, characterization, crystal structure and cytotoxicity
of 2,4-bis(selenomethyl)quinazoline
•
•
•
´
Daniel Plano Elena Iban˜ez Juan Antonio Palop
´
Carmen Sanmartın
Received: 18 January 2011 / Accepted: 31 May 2011 / Published online: 17 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Organoselenium compounds have already been
reported to be good anticarcinogenic candidates. A new
selenoquinazoline derivative, 2,4-bis(selenomethyl)qui-
nazoline (compound 1), has been synthesized, spectro-
scopically characterized and its crystal structure has been
studied. An intermolecular coupling between C2 and H05 in
the Heteronuclear Multiple Bond Correlation (HMBC)
experiment has been observed. Assuming that the head-
to-tail overlap of parallel molecules (as identified by X-ray
diffraction) remains in solution to give bimolecular enti-
ties, the p–p interaction enables heteronuclear coupling
between the former atoms with a three-bond distance
[C2ꢀꢀꢀ(p–p)ꢀꢀꢀC05–H50 ]. The crystal structure of compound 1
has been solved by X-ray diffraction. It crystallizes in tri-
clinic system, space group P-1. Unit cell parameters are
Introduction
Selenium (Se) is an essential trace element that is involved
in different physiological functions in the human body.
Epidemiological and clinical studies indicate that inade-
quate levels of selenium increase the risk of cancer and
nutritional intervention studies show that high Se intakes
effectively reduce mammary, prostate, lung, colon and
liver cancer risk [1–5]. Basic research and clinical trials
strongly support the protective role of selenium against
various types of cancer. This effect was more remarkable
in prostate and colorectal cancers [6–8]. A recent report
from the Selenium and Vitamin E Cancer Prevention Trial
(SELECT) demonstrated that selenium or vitamin E, either
alone or in combination did not prevent prostate cancer or
secondary cancers [9]. However, the failure of one form of
Se does not disprove the possible protective effect of other
forms of Se. In fact, the anticancer activity of Se is
dependent on its chemical form and dosage. The results of
the SELECT caused much controversy, the formulation of
Se used (selenomethionine) being one of the topics dis-
cussed [10]. Selenomethionine can be incorporated non-
specifically into proteins in place of methionine [11].
Compartmentation into tissue proteins limits selenomethi-
onine from being further metabolized.
˚
˚
˚
a = 7.4969(7) A, b = 8.7008(8) A, c = 10.1666(9) A,
a = 110.215(2)°, b = 90.354(2)°, c = 115.017(1)°. Linear
chains in crystals of compound 1 are generated by
C–HꢀꢀꢀSe and SeꢀꢀꢀSe bonds between molecules. Further-
more, head-to-tail overlap of parallel molecules, in which
p–p interactions can occur, is observed. Compound 1
exhibited a cytotoxic effect in all of the evaluated tumoral
cell lines and showed a higher cytotoxic effect in colon and
breast cancer cell lines than etoposide, which was used as a
reference compound.
The mechanisms of action for Se and selenocompounds
as anticancer agents are not fully understood. However, it
has been proposed in the literature that antioxidant prop-
erties [12], cell cycle modulation [13, 14], apoptosis
induction [15, 16] and angiogenesis inhibition [17, 18] may
be some of the possible molecular targets for Se
compounds.
Keywords Selenium ꢀ Synthesis ꢀ
Spectroscopic characterization ꢀ Crystal structure ꢀ
X-ray diffraction ꢀ Cytotoxicity
´
´
D. Plano (&) ꢀ E. Iban˜ez ꢀ J. A. Palop ꢀ C. Sanmartın
Department of Organic and Pharmaceutical Chemistry,
University of Navarra, Irunlarrea, 1, 31008 Pamplona, Spain
e-mail: dplano@alumni.unav.es
One of our ongoing research projects involves the
investigation of novel potential antitumor and leishmani-
cidal agents containing at least one selenium atom [19–24].
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Struct Chem (2011) 22:1233–1240
Synthesis of 2,4-dichloroquinazoline
mixture of 2,4(1H,3H)-quinazolinedione [2.00 g;
Our research group has synthesized a series of imidosele-
nocarbamates, some of which show better in vitro antitu-
mor effects than certain chemotherapeutic agents
(including etoposide, taxol and methotrexate) in several
cell lines [19, 20]. These derivatives also showed antile-
ishmanial activity in vitro against Leishmania infantum
[21]. Our research group also prepared and evaluated a
series of selenylacetic acids [22], selenocyanates and
diselenides derivatives [23], and selenadiazoles [24].
The work described here involved the synthesis and
cytotoxicity evaluation of 2,4-bis(selenomethyl)quinazo-
line (1) in four human tumour cell lines (CCRF-CEM,
HTB54, HT-29 and MCF-7) and one non-malignant
mammary gland-derived cell line (184B5). In addition, we
present the molecular characterization and the X-ray
crystal structure determination for this compound.
A
12.3 mmol] in a large excess of phosphoryl chloride
[10 mL] and N,N-dimethylaniline [1.6 mL; 12.5 mmol]
was heated under reflux for 14 h. The mixture was poured
onto ice [200 g] and the resulting solid was filtered off,
washed with distilled water and dried. This compound was
used without further purification.
Synthesis of quinazoline-2,4-diselenol hydrochloride
Selenourea [1.25 g; 10.2 mmol] was added to a solution of
2,4-dichloroquinazoline [1.85 g; 9.3 mmol] in absolute
ethanol [50 mL] and the reaction mixture was heated under
reflux for 2 h. The resulting solid was filtered off while
warm and washed with distilled water and cold ethanol
[3 9 25 mL]. The desired compound was obtained and
used without further purification.
Experimental
M = 288.07 g mol-1
.
Yield: 83%. m.p. 228.7
1.4 °C. IR (KBr pellet, cm-1): 1610 [m(C2=N)], 1552
Synthesis and characterization
[m(C4=N)]. 1H-NMR (400 MHz, DMSO-d6) (d/ppm):
7.34–7.41 (m, 2H, H5 ? H7); 7.85 (t, 1H, J6-7
=
Melting points were determined with
a
Mettler
J6-5 = 7.7 Hz, H6); 8.30 (t, 1H, J8-7 = 8.2 Hz, H8); 13.84
(bs, 2H, 2SeH). Anal Calcd. for C8H6N2Se2 (0.5 HCl): C
31.34, H 2.12, N 9.14. Found: C 31.20, H 1.96, N 9.15.
FP82 ? FP80 apparatus (Greifense, Switzerland) and are
uncorrected. The 1H-NMR spectra were recorded on a
Bruker 400 UltrashieldTM spectrometer (Rheinstetten,
Germany) using TMS as the internal standard. The IR
spectra were obtained on a Thermo Nicolet FT-IR Nexus
spectrophotometer with KBr pellets. Elemental microa-
nalyses were carried out on vacuum-dried samples using a
LECO CHN-900 Elemental Analyzer. AlugramÒ SIL
G/UV254 (Layer: 0.2 mm) (Macherey–Nagel GmbH &
Co. KG. Postfach 101352, 52313 Du¨ren, Germany) was
used for Thin Layer Chromatography. Chemicals were
purchased from E. Merck (Darmstadt, Germany), Scharlau
Synthesis of 2,4-bis(selenomethyl)quinazoline (1)
Iodomethane [1.57 mL; 25.2 mmol] was added to a solu-
tion of quinazoline-2,4-diselenol hydrochloride [2.57 g;
8.4 mmol] in 0.4 N NaOH [20 mL]. The mixture was
stirred at room temperature for 1 h. The resulting solid was
filtered off, washed with distilled water and recrystallized
from ethanol.
M = 316.12 g mol-1. Yield: 80%. m.p. 61.4 0.8 °C.
IR (KBr pellet, cm-1): 1608 [m(C2=N)], 1552 [m(C4=N)].
1H-NMR (400 MHz, DMSO-d6) (d/ppm): 2.53 (s, 3H,
´
(F.E.R.O.S.A., Barcelona, Spain), Panreac Quımica S.A.
(Montcada i Reixac, Barcelona, Spain), Sigma-Aldrich
´
Quımica S.A. (Alcobendas, Madrid, Spain), Acros Organ-
Se41–CH3); 2.56 (s, 3H, Se21–CH3); 7.61 (ddd, 1H, J6-5
=
¨
ics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Belgie) and
8.2 Hz, J6-7 = 7.0 Hz, J6-8 = 1.2 Hz, H6); 7.77 (dd, 1H,
J8-7 = 8.4 Hz, H8); 7.92 (ddd, 1H, J7-5 = 1.2 Hz, H7);
7.95 (dd, 1H, H5). 13C-NMR (100 MHz, DMSO-d6) (d/
ppm): 6.7 (Se41–CH3); 7.6 (Se21–CH3); 124.2 (C10); 126.1
(C5); 127.8 (C6 ? C8); 135.7 (C7); 148.7 (C9); 164.6 (C4);
171.8 (C2). Anal Calcd. for C10H10N2Se2: C 37.97, H 3.16,
N 8.86. Found: C 37.86, H 3.03, N 8.72.
Lancaster (Bischheim-Strasbourg, France).
Chemical preparation
The synthetic route for 2,4-bis(selenomethyl)quinazoline
(1) is depicted in Fig. 1.
Fig. 1 Synthetic route for
preparation of 2,4-
bis(selenomethyl)quinazoline
(1)
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Struct Chem (2011) 22:1233–1240
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X-ray structure determination
2 mM L-glutamine, 100 units/mL penicillin, 100 lg/mL
streptomycin and 10 mM HEPES buffer (pH 7.4). MCF-7
cells were grown in EMEM medium (Clonetics) supple-
mented with 10% foetal calf serum, 2 mM L-glutamine,
100 units/mL penicillin and 100 lg/mL streptomycin.
184B5 cells were grown in Hams F-12/DMEM (50:50)
supplemented as described in the literature [27]. Cytotox-
icity was then determined by the MTT method. Results are
expressed as GI50, the concentration that reduces by 50%
the growth of treated cells with respect to untreated con-
trols, TGI, the concentration that completely inhibits cell
growth, and LC50, the concentration that kills 50% of the
cells. Data were obtained from at least 3 independent
experiments performed in quadruplicate.
Intensity data were collected on a Bruker SMART-APEX
automatic X-ray diffractometer with a CCD area detector
using graphite-monochromated Mo Ka radiation (k =
˚
0.71073 A). A trial data collection revealed that the
selected crystal was of high quality and this permitted the
measurement of preliminary unit cell parameters. The
structure determination was carried out by collecting dif-
fracted intensities through 1365 exposures/scans with 10 s/
exposure to scan in Ewald half-sphere at least 25° of h
completely. Absorption corrections (program SADABS
[25]) were applied to the intensity data. SHELXTL-NT 6.1
[26] was employed to solve and refine the crystal structure.
The structure was refined by the least-squares method on
F2 using all reflections. The non-hydrogen atoms were
refined with anisotropic displacement factors. The posi-
tions of hydrogen atoms were calculated considering the
nature of the atom that they are linked to. The
(x,y,z) coordinates of these hydrogens were refined jointly
with the atoms to which they are bonded and isotropic
displacement parameters set at 1.5 times (methylic hydro-
gens) or 1.2 times (benzylic hydrogens) the Ueq of the
parent atoms were used.
Results and discussion
Molecular characterization
The m(C=N) of the quinazoline moiety in the IR spectrum
can be assigned to bands at 1608 and 1552 cm-1. The first
of these bands could correspond to a C2=N bond because
C2 supports a higher positive charge density than C4 since
it is located between two nitrogen atoms. Therefore, a
stronger bond exists for the C2 atom and the corresponding
band will appear at higher wavenumber than that for C4.
The structure of compound 1 could not be unequivocally
assigned by monodimensional NMR experiments and a
bidimensional experiment was therefore carried out. The
aims of the HMBC experiment were twofold: (a) the cor-
rect assignment of the signals for the methyl groups
The space group determination is not unequivocal, since
for a triclinic lattice two space groups are possible: P1 and
P-1. Space group P-1 seems to be more probable
according to the distribution of intensities and other sta-
tistical parameters. The resolution was carried out by direct
methods. All non-hydrogen atoms in the molecule were
found using an E-map prepared from the set of stages with
the best figure of merit, which has confirmed the space
group.
1
attached to the selenium atoms in the H-NMR spectrum;
and (b) the unequivocal assignment of the signals of the
aromatic carbon atoms in the 13C-NMR spectrum.
Cytotoxicity
It can be seen from the HMBC spectrum shown in Fig. 2
that there is heteronuclear coupling between the protons of
the methyl group attached to Se21 and the C2 atom
(2.56–171.8 ppm). The same coupling appears between the
protons of the methyl group attached to Se41 and the C4 atom
(2.53–164.6 ppm). The assignment of the aromatic carbon
signals was made based on the coupling observed with the
aromatic hydrogen signals. Furthermore, an unexpected
coupling between H5 and C2 (7.95–171.8 ppm) was
observed. This signal might not correspond to an intramo-
lecular coupling due to the five-bond distance between these
two atoms. A possible explanation for this observation was
investigated by single crystal X-ray diffraction.
The cytotoxic effect of compound 1 was tested at five
different concentrations between 0.01 and 100 lM. Com-
pound 1 was initially dissolved in DMSO at a concentra-
tion of 0.1 M and serial dilutions were prepared using
culture medium. The plates with cells from the different
lines, to which medium containing the substance under test
was added, were incubated for 72 h at 37 °C in a humid-
ified atmosphere containing 5% CO2. Human tumour cell
lines were provided by the European Collection of Cell
Cultures (ECACC) or the American Type Culture Collec-
tion (ATCC). Five cell lines were used: one human lym-
phocytic leukaemia (CCRF-CEM), three human solid
tumours, one colon carcinoma (HT-29), one lung carci-
noma (HTB-54), one breast adenocarcinoma (MCF-7) and
one non-tumoral cell line (184B5). CCRF-CEM, HT-29
and HTB-54 cells were grown in RPMI 1640 medium
(Invitrogen) supplemented with 10% foetal calf serum,
Crystal characterization
The crystal data and refinement details for compound 1 are
listed in Table 1. The optimization of the unit cell
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Struct Chem (2011) 22:1233–1240
Fig. 2 HMBC spectrum of
compound 1
parameters was performed by least-squares refinement
of the angular positions of the 1692 reflections in the
range from 2.80° to 27.69° (h). These parameters corre-
spond to a triclinic lattice. The adjustment by a least-
squares method of the angular positions of 1674 reflections
in the range from 2.81° to 27.61° (h) considering the
cell as pseudomonoclinic led to the following parameters:
Evidence for weak intermolecular hydrogen bonds
Car–HꢀꢀꢀSe is observed in the crystal structure:
˚
˚
C6–H6, 0.93 A; C6ꢀꢀꢀSe21(i), 3.866(5) A; H6ꢀꢀꢀSe21(i),
˚
3.05 A; C6–H6ꢀꢀꢀSe21(i), 147° [(i): x, y, z ? 1].
˚
˚
C8–H8, 0.93 A; C8ꢀꢀꢀSe41(i), 3.998(5) A; H8ꢀꢀꢀSe41(i),
˚
3.14 A; C8–H8ꢀꢀꢀSe41(i), 155° [(i): x, y, z ? 1]
The first such bond leads to infinite molecular chains in
the [010] direction (Fig. 4a). On the other hand, there are
SeꢀꢀꢀSe contacts [Se41ꢀꢀꢀSe41(-x ? 1, -y ? 1, -z ? 1)]
˚
˚
˚
a = 15.7692(14) A, b = 7.4969(7) A, c = 10.1652(10) A,
b = 112.593(2)° and the a and c parameters deviated sig-
nificantly from 90°: 90.326(2)° and 89.524(2)°, respec-
tively. Nevertheless, analysis of the equivalent reflections,
considering the crystal as monoclinic, revealed an Rint
value of 0.54, which indicates that the cell is not
monoclinic.
˚
with a distance, 3.7399(6) A, that is shorter than the sum of
˚
the van der Waals radii, 3.80 A (Fig. 4b).
Moreover, head-to-tail overlap of parallel molecules, in
which p–p interactions can occur, is observed (Fig. 5a).
˚
The distance between the molecular planes is 3.5 A and the
A representation of the molecular structure based on the
X-ray analysis, showing thermal ellipsoids is shown in
Fig. 3. Table 2 includes geometric parameters of com-
pound 1.
normal component to these planes shows an angle of 23°
with the stacking direction. Data show that the distance
between the pyrimidine ring centroids and the distance
between the benzene centroid and the pyrimidine centroid
of adjacent molecules in the stack is almost the same,
˚
3.814(2) A, while the distance between the benzene cen-
˚
troids is 5.102(2) A (Fig. 5b).
The direction of the C4–Se41 bond coincides with the
corresponding cyclic angle bisector, N3–C4–C10, while the
position of the C2–Se21 bond is slightly asymmetric, dis-
tanced from the other substituent [exocyclic angles of
113.6° and 117.5(2)°] (Table 2).
Considering the arrangement of the two molecules in a
stack (Fig. 5b), a possible explanation for the coupling
between H5 and C2 seen in the HMBC experiment (Fig. 2)
can be proposed. Assuming that the head-to-tail overlap of
parallel molecules remains in solution to give bimolecular
entities, the p–p interaction enables the heteronuclear
coupling between the former atoms with a three-bond
The quinazoline unit is almost flat and the largest devia-
˚
tion from the plane defined by the ten atoms is 0.012(3) A.
The two substituents are located practically in the mean
plane of the quinazoline unit with the methyl groups close to
it (i.e. forming the shape of a clamp, Fig. 3).
123