Novel DNA fluorescence probes based on 2-thioxo-tetrahydro-4H-imidazol-4- ones: Synthetic and biological studies

Article abstract of DOI:10.1016/j.tetlet.2011.10.118

An efficient method for the liquid-phase synthesis of 3,5-disubstituted thiohydantoins has been developed. This new class of substituted thiohydantoins was tested as DNA fluorescence probes. Anthracene-substituted thiohydantoin undergoes a change in fluor

Full text of DOI:10.1016/j.tetlet.2011.10.118

Tetrahedron Letters 53 (2012) 51–53
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Novel DNA fluorescence probes based on 2-thioxo-tetrahydro-4H-imidazol-4-
ones: synthetic and biological studies
⇑
Alexander G. Majouga , Anna V. Udina, Elena K. Beloglazkina, Dmitrii A. Skvortsov, Maria I. Zvereva,
Olga A. Dontsova, Nikolay V. Zyk, Nikolay S. Zefirov
Chemistry Department of Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2011
Revised 10 October 2011
Accepted 21 October 2011
Available online 28 October 2011
An efficient method for the liquid-phase synthesis of 3,5-disubstituted thiohydantoins has been devel-
oped. This new class of substituted thiohydantoins was tested as DNA fluorescence probes. Anthra-
cene-substituted thiohydantoin undergoes a change in fluorescence in the presence of DNA.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Thiohydantoin
DNA probes
Fluorescence
In recent years the development of fluorescent probes which
provide information in various fields has attracted much atten-
tion.1–3 The strong influence of the surrounding medium on fluo-
rescence emission has led to fluorescent molecules being used as
probes for the investigation of physicochemical, biochemical and
biological systems. Studies on the interactions between DNA and
ligands are particularly important for therapeutic4 and scientific
reasons.5 Low molecular weight fluorescent organic compounds
which are able to bind nucleic acids, are attractive in biotechnology
as the basis for fluorescent DNA and RNA. Such dyes are widely
used in molecular biology, genetic engineering and some medical
tests (genetic analysis, tests for pathogens, etc.). The most com-
monly used dye is ethidium bromide. It is an intercalating agent
which is toxic and may be mutagenic. Some stains, such as SYBR
Green and SYBR Gold are safer and can bind preferentially to one
of the conformations of DNA. These dyes may change the electro-
phoretic mobility of nucleic acids.6 However SYBR is expensive
and sensitive to light. The formation of new, highly-selective and
inexpensive stains for nucleic acids is therefore an important
target.
stains. Extensively studied DNA intercalators include ethidium
bromide, proflavine, daunomycin, doxorubicin and thalidomide.
These compounds are used in chemotherapeutic treatment to inhi-
bit DNA replication in rapidly growing cancer cells. Examples in-
clude doxorubicin (adriamycin) and daunorubicin, both of which
are used in the treatment of Hodgkin’s lymphoma, and dactinomy-
cin, which are used in Wilm’s tumour, Ewing’s sarcoma and
rhabdomyosarcoma.
Bearing in mind earlier observations and combined with our
current research, thiohydantoin (2-thioxo-tetrahydro-4H-imida-
zol-4-one) derivatives are good candidates for DNA probes. These
compounds have a rigid planar structure and a number of hetero-
atoms that may form hydrogen bonds. For the fluorescent moiety
we studied fluorescein, naphthalene and anthracene derivatives.
2-Thiohydantoins were synthesized by the condensation of
3-substituted thiones with heteroaromatic aldehydes, such as 2-
pyridinecarboxaldehyde and 2-quinolinecarboxaldehyde in basic
medium (Scheme 1, Table 1).9 5-Hetaryl substituted thiohydanto-
ins were synthesized in good yields and existed as Z-isomers, as
established from the chemical shift of the vinyl proton. Alkylation
of compounds 1a–h with methyl iodide resulted in the formation
of S-alkylated products 2a–g.10,11
Compounds with rigid and planar structures are good candi-
dates for DNA intercalation.7,8
Several ligands interact with DNA through covalent and electro-
static binding or intercalation. Intercalation occurs when ligands of
a suitable size and chemical nature fit themselves in between base
pairs of DNA. These ligands are usually polycyclic, aromatic and
planar structures, and consequently often make good nucleic acid
Primary screening (Fig. 1) did not reflect exactly the fluores-
cence changes in the presence of DNA, because DNA sodium salts
change the pH, which may result in the modification of fluores-
cence. Spectroscopic measurements were made in the presence
of the buffer, TBE 1ꢀ, (pH 8.2) to eliminate this possible effect.
We decided to test substrate 1c, as it is not fluorescein-based (fluo-
rescein is sensitive to pH), but alters its fluorescence in the
presence of DNA.
⇑
Corresponding author. Tel.: +7 925 858 1024; fax: +7 495 743 9082.
E-mail address: majouga@org.chem.msu.ru (A.G. Majouga).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.118

52
A. G. Majouga et al. / Tetrahedron Letters 53 (2012) 51–53
R1
N
R1
N
R1
N
O
S
O
S
1) KOH, EtOH
CH3I, KOH
EtOH, H2O
Me
O
S
R2
CHO
N
N
N
2) dilute HCl
H
H
R2
R2
2
1
Scheme 1. Synthesis of 3,5-disubstituted thiohydantoins.
Table 1
Structures of the of 3,5-disubstituted thiohydantoins prepared
Product R1
R2
Yield (%)
90
1a
N
N
1b
1c
89
92
Figure 1. Primary screening of fluorescence using a UV transilluminator (excitation
frequency of band with a maximum at 312 nm) at different DNA concentrations in
aqueous solution.
N
N
HO
O
OH
We recorded synchronous spectra of 1c with gaps of 50 and
30 nm to determine whether and where the differences in the
presence of DNA occurred (spectra with a 30 nm gap are shown
in Fig. 2A). It was found that in the presence of DNA the fluores-
cence increased when the excitation wavelength was about
490 nm (the fluorescence reduction at 250–300 nm was due to
the absorption by DNA). We verified this observation using the
fluorescence spectra of 1c with an excitation at 495 nm. The fluo-
rescence increased (515 nm peak) in the presence of DNA
(0.5 mg/ml) up to threefold (Fig. 2B).12
Substance 1c is a new compound which changes its fluores-
cence in the presence of DNA. It may be speculated that it specifi-
cally interacts with DNA through the anthracene moiety as has
been shown previously.13
In this Letter, substituted 2-thioxo-tetrahydro-4H-imidazol-4-
one derivatives were synthesized and characterized by IR, MS, 1H
NMR and elemental analysis. A novel fluorescent reagent 1c was
synthesized and tested in model DNA binding experiments. Further
O
1d
65
O
1e
1f
Pr
84
83
88
N
N
N
All
Ph
1g
Figure 2. (A) Synchronous spectra for 1c (16 ll) with a 30 nm gap in the presence of DNA (0.1 or 0.5 mg/ml). (B) Fluorescence spectra with excitation at 495 nm for 1c in the
presence of DNA (0.1 or 0.5 mg/ml).

A. G. Majouga et al. / Tetrahedron Letters 53 (2012) 51–53
53
Science and Education (contract No. 16.740.11.0203, G1390) is
Table 1 (continued)
gratefully acknowledged.
Product R1
R2
Yield (%)
O
OH
HO
References and notes
N
1. Neuhaus, W.; Trzeciak, J.; Lauer, R.; Lachmann, B.; Noe, C. R. J. Pharm. Biomed.
Anal. 2006, 40, 1035–1039.
2. Sevestre, A.; Charmantray, F.; Hélaine, V.; Lásiková, A.; Hecquet, L. Tetrahedron
2006, 62, 3969–3976.
O
1h
74
O
3. Gonçalves, M. S. T. Chem. Rev. 2009, 109, 190–212.
4. Lippard, S. J. Acc. Chem. Rev. 1978, 11, 211–217.
5. Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933–
8938.
6. Huang, Q.; Fu, W. L. Clin. Chem. Lab. Med. 2005, 43, 841–842.
7. Juewen, L.; Zehui, C.; Yi, L. Chem. Rev. 2009, 109, 1948–1998.
8. Meyer-Almes, F. J.; Porschke, D. Biochemistry 1993, 32, 4246–4253.
9. General procedure for the synthesis of compounds 1a–h: 3-substituted-2-
thioxotetrahydro-4H-imidazol-4-one (1 equiv) was dissolved in a 2% ethanolic
solution of KOH. After complete dissolution an equivalent of hetaryl
carbaldehyde was added. The mixture was stirred for 12 h and neutralized
with HCl. The resulting precipitate was crystallised from EtOH/DMF.
Compound 1a was synthesized from 3-(1-naphthyl)-2-thioxoimidazolidin-4-
one (4 g, 15 mmol) and 2-pyridinecarboxaldehyde (1.76 g, 16 mmol) in 90%
yield (m.p. 205 °C): 1H NMR (400 MHz, DMSO-d6) d 6.89 (s, 1H, CH@), 7.44 (dd,
2a
85
69
N
2b
N
N
HO
O
OH
J1 = 5.7 Hz, J2 = 2.5 Hz, 1H, H -Py), 7.60 (m, 5H, Ar), 7.83 (d, J = 7.8 Hz, 1H, Hb-
O
c
2c
70
0
Py), 7.95 (t, J = 7.7 Hz, 1H, Hb -Py), 8.07 (d, J = 8.0 Hz, 1H, Ar), 8.12 (d, J = 7.7 Hz,
1H, Ar), 8.84 (d, J = 4.2 Hz, 1H, H -Py), 12.01 (br s, 1H, NH); IR (cmꢁ1, KBr):
0
a
O
3330, 1740, 1600.
10. (a) Beloglazkina, E. K.; Majouga, A. G.; Romashkina, R. B.; Zyk, N. V. Tetrahedron
Lett. 2006, 47, 2957–2959; (b) Beloglazkina, E. K.; Majouga, A. G.; Romashkina,
R. B.; Moiseeva, A. A.; Zyk, N. V. Polyhedron 2007, 26, 797–802; (c) Ermoli, A.;
Bargiotti, A.; Brasca, M. G.; Ciavolella, A.; Colombo, N.; Fachin, G.; Isacchi, A.;
Menichincheri, M.; Molinari, A.; Montagnoli, A.; Pillan, A.; Rainoldi, S.; Sirtori,
F. R.; Sola, F.; Thieffine, S.; Tibolla, M.; Valsasina, B.; Volpi, D.; Santocanale, C.;
Vanotti, E. J. Med. Chem. 2009, 52, 4380–4390.
2d
2e
2f
Pr
79
74
72
N
N
N
N
All
11. General procedure for the synthesis of compounds 2a–g: Compound 1a, 1c–h
(1 equiv) and MeI (1.1 equiv) were suspended in H2O/EtOH (1:1) mixture. A
solution of KOH (1 equiv in H2O) was slowly added to this mixture at room
temperature. After 2 h the resulting solid was collected by filtration and
washed with EtOH. After drying under vacuum, analytically pure samples were
obtained. Compound 2a was prepared from 1a (2 g, 6 mmol) and MeI (0.852 g,
6 mmol) in 85% yield (m.p. 224 °C): 1H NMR (400 MHz, CDCl3) d 2.73 (s, 3H,
0
Ph
HO
O
OH
CH3), 6.65 (m, 8H, Hb -Py, Hb-Py, H -Py+Ar, CH@), 7.15 (d, J = 7.5 Hz, 1H, Ar),
c
0
7.35 (d, J = 8.2 Hz, 1H, Ar), 7.63 (m, 2H, H -Py+Ar), 7.99 (d, J = 7.0 Hz, 1H, Ar);
a
IR (cmꢁ1, KBr):1740, 1670, 1600; Elemental analysis: calcd (%): C 69.54, H 4.38,
N 12.16. C20H15N3OS; found: C 69.34, H 4.20, N 12.55.
O
2g
65
12. DNA fluorescence screening: A mixture of H2O (52
ll), substrate (8 ll) in DMSO
(0.1; 1; 10 mM) and 20 l of 1 mg/ml DNA from calf thymus aqueous solution
l
O
was prepared. The mixture was shaken, incubated at room temperature for
30 min and centrifuged for 5 min at 16,000 g, transferred onto a new plate and
photographed with a Chemidoc (BioRad). Synchronous spectra were taken
with gaps of 50 and 30 nm. The fluorescence spectra were recorded at the
excitation wavelengths determined from the synchronous spectra.
ˇ
13. Kozurkováa, M.; Sabolováa, D.; Paulíkováb, H.; Janovec, L.; Kristianc, P.;
investigations are needed in order to understand the interaction
mechanism and fluorescence effects.
´
Bajdichováb, M.; Bušad, J.; Podhradskya, D.; Imrichc, J. Int. J. Biol. Macromol.
2007, 41, 415–422.
Acknowledgments
Financial support of this work by the Russian fund for basic re-
search (Grant No. 10-03-00-677), Russian Federation Ministry of