The synthesis and spectral properties of new DNA binding ligands

Article abstract of DOI:10.1016/j.tetlet.2010.07.180

Two new ligands based on anthracene or carbazole planar skeletons, and a phenyloxazoline moiety linked by a vinyl bridge are synthesized as potential DNA-interacting drugs. Their spectral characteristics and DNA binding affinity are assessed.

Full text of DOI:10.1016/j.tetlet.2010.07.180

Tetrahedron Letters 51 (2010) 5415–5418
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
The synthesis and spectral properties of new DNA binding ligands
⇑
´
´
Agata Głuszynska , Kamila Bajor, Izabella Czerwinska, Dominika Kalet, Bernard Juskowiak
´
Laboratory of Bioanalytical Chemistry, Faculty of Chemistry, A. Mickiewicz University, Poznan 60-780, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new ligands based on anthracene or carbazole planar skeletons, and a phenyloxazoline moiety
linked by a vinyl bridge are synthesized as potential DNA-interacting drugs. Their spectral characteristics
and DNA binding affinity are assessed.
Received 15 June 2010
Revised 21 July 2010
Accepted 30 July 2010
Available online 6 August 2010
Ó 2010 Published by Elsevier Ltd.
Small organic molecules (ligands) can interact selectively with
DNA via the minor groove, or by intercalation, but the interaction
preferences depend on the ligand structure and on the nature of
DNA. Small differences in the structure of a DNA-interacting ligand
may affect the binding preferences and stability of the ligand/DNA
complex.1,2
G-Quadruplexes are structural forms of nucleic acids and con-
tain G quartets (each consists of four guanines held together by
eight hydrogen bonds). These tetraplex structures are stabilized
in the presence of specific metal cations (Na+, K+). With hypotheses
that G-quadruplex structures play an important role in biological
processes, the level of interest in these structures has increased.
The ligands which selectively interact with the G-quadruplex are
called ‘G-quadruplex ligands’, and are known to exhibit anticancer
activities and their number has grown rapidly over the past few
years.2–5
Our research group is interested in the synthesis of ligands
containing anthracene, and carbazole skeletons, which can bind
to the secondary structure of guanine-rich DNA. The designed
new ligands 1, and 2, are expected to interact with the G-quadru-
plex via external stacking due to the presence of a planar anthra-
cene or carbazole unit and a vinyloxazoline arm coupled with
delocalized
p-electrons. The polyaromatic moieties may exhibit
steric effects, and photochemical activity, and are reported to pro-
mote selectively the formation and/or stabilization of higher-order
DNA structures.6 The structures of the ligands can be easily altered
by cis–trans photoisomerization of a double bond.7 Finally, the
oxazoline moiety may serve as a potential donor of electrons,
and can be installed as a chiral non-racemic group.8 The ligands
1, and 2 were synthesized via the Arbuzov–Horner modification
of the Wittig reaction (Scheme 1).
The starting diethyl[(4-cyanophenyl)methyl]phosphonate was
prepared from p-toluonitrile by known synthetic procedures in
79% overall yield.9,10 The 9-cyano-10-anthracenocarboxaldehyde
(5) was prepared from 9,10-dibromoanthracene (3) in two
Scheme 1. Reagents and conditions: (i) (1) n-BuLi, THF, ꢀ72 °C, 1 h; (2)
4-formylmorpholine, ꢀ35 °C, 12 h; (ii) CuCN (10 equiv), DMSO, 190 °C, 6 h; (iii)
CNC6H4CH2PO(OC2H5)2, MeONa, DMF, 0 °C, 3 h; (iv) CNC6H4CH2PO(OC2H5)2,
MeONa, DMF, reflux, 22 h; (v) ( )-1-amino-2-propanol (40 equiv), ethylene gly-
col/glycerol, K2CO3, 115 °C, 72 h; (vi) ( )-1-amino-2-propanol (10 equiv), ethylene
glycol/glycerol, K2CO3, 115 °C, 22 h.
steps.11–13 Addition of n-buthyllithium to 3 was carried out follow-
ing the general reaction conditions specific for this type of reaction.
The best results in terms of chemical yield were achieved using
N-formylmorpholine as a formylating agent.14 9-Bromoanthra-
cene-10-carboxaldehyde (4) was reacted with copper(I) cyanide
in DMSO to afford 5 in a good yield of 81%,15 while in DMF the
⇑
Corresponding author. Tel.: +48 61 8291467; fax: +48 61 8658008.
E-mail address: aglusz@amu.edu.pl (A. Głuszyn´ ska).
0040-4039/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2010.07.180

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A. Głuszynska et al. / Tetrahedron Letters 51 (2010) 5415–5418
5416
reaction product was not observed.16 Application of the Arbuzov–
Horner modification of the Wittig reaction using MeONa as base in
DMF afforded the expected product in moderate yields.17 With t-
BuOK no product was observed.18 Nitrile 8 was obtained from car-
bazole 6 in a similar manner.
Nitriles 7, and 8,19 possessing almost exclusively the E,E-config-
uration, as indicated by 1H NMR spectroscopic analysis, were con-
verted into oxazolines 1, and 2,20 by known synthetic procedures
in moderate yields.21 In the case of compound 7 with two nitrile
groups (IR: 2224 cmꢀ1 (C6H4–C„N) and 2211 cmꢀ1 (C14H8–
C„N), only the benzylic nitrile underwent conversion to an oxaz-
oline moiety (disappearance of the band at 2224 cmꢀ1).
The new ligands 1, and 2 are soluble in several organic solvents
and the UV/Vis absorption spectra did not depend on the nature of
the solvent (some changes in intensity of the absorption bands oc-
curred without remarkable band shifts) as shown in Table 1.
Small solvent-dependent shifts (1–8 nm) and variations in mo-
lar absorptivities were observed, but they did not seem to have any
correlation with the polarity of the solvents. In the case of ligand 1
a broad band at ca. 413 nm suggests conjugation between the an-
thryl chromophore and the phenyl-oxazoline system.
In contrast to organic solvents, ligands 1, and 2 were only par-
tially soluble in water, and aqueous buffer solution. The absorption
spectra of aqueous solutions of the ligands, prepared by injection
of a small volume of ligand stock solution in EtOH, showed a grad-
ual decrease in absorbance with time. The same effects were ob-
served in the fluorescence spectra of aqueous solutions, which
indicated aggregation phenomena, and slow precipitation pro-
cesses. Indeed, after 30 min, the absorbance decreased by ca.
30%, showing a noticeable turbidity Fig. 1).
Since ligands 1, and 2 were designed as potential DNA binding
agents, their tendency for aggregation in aqueous solutions made
further studies difficult. On the other hand, DNA preserves its high-
er-order structure in the presence of moderate amounts of organic
solvents such as EtOH or DMSO (up to 10%). Therefore, we investi-
gated the spectral properties of the ligands in mixed H2O/EtOH sys-
tems containing 5–80% EtOH. Satisfactory spectral stability was
observed in all H2O/EtOH mixtures, even solutions containing only
5% EtOH exhibited good stability, and reproducibility of spectral
parameters.
The absorbance of ligands 1 and 2 in 5% EtOH was noticeably
lower than those in pure EtOH, but they did not change with time.
It should be noted that for both ligands, molar absorptivities in 5%
EtOH solution were remarkably lower than those in EtOH or other
organic solvents (Table 1).
The fluorescence spectra of the ligands were recorded in EtOH
and in H2O/EtOH-mixed solvent systems. The most interesting re-
sults are shown in Figure 2. The ligand 2 exhibits a broad weak
emission band at 477 nm, and at 487 nm in 96% EtOH, and 5%
EtOH, respectively, but the latter is slightly red-shifted and broad-
ened. These effects and the much lower molar absorptivity may
Figure 1. Absorption spectra of ligand 1 (A): (1.9 ꢁ 10ꢀ5 M) and ligand 2 (B):
(4.3 ꢁ 10ꢀ5 M) in EtOH and 5% EtOH.
2 in EtOH
2 in 5%EtOH
1 in EtOH
600
1 in 5%EtOH
400
200
0
400
450
500
550
600
650
700
Wavelength (nm)
Figure 2. Fluorescence emission spectra of ligand 1 (1.19 ꢁ 10ꢀ5 M in 96% EtOH
(green) and 5% EtOH (pink), kex = 388 nm), and ligand 2(1.5 ꢁ 10ꢀ5 M in 96% EtOH
(black) and 5% EtOH (red), kex = 350 nm).
Table 1
Effect of solvent on the spectral characteristics of the absorption spectra of ligands 1
and 2
ꢁ 10ꢀ4/Mꢀ1 cmꢀ1
)
indicate the formation of less fluorescent dimers in aqueous solu-
tion containing only 5% EtOH.
Solvent
kmax/nm (
Ligand 1
e
Ligand 2
Interestingly, the fluorescence spectra of ligand 1 exhibited
more complex behavior depending on the mixed solvent composi-
tion. Bimodal fluorescence was observed in 96% EtOH with one
emission band at ca. 450 nm, and a second band at ca. 550 nm.
The first band shows vibronic structure characteristic of the
anthracene emission and may represent the monomer fluores-
cence whereas the broad long-wavelength fluorescence originates,
probably from the excited dimer of ligand 1. These assignments
MeOH
EtOH
5% EtOH
CH3CN
CHCl3
411 (1.61)
412 (1.72)
424 (0.58)
412 (1.66)
413 (1.66)
419 (1.54)
415 (1.61)
260 (12.7)
260 (12.9)
266 (2.47)
260 (10.5)
263 (10.2)
nda
356 (3.3)
358 (3.3)
363 (1.27)
355 (3.4)
357 (3.2)
359 (2.8)
358 (3.4)
239 (3.0)
237 (2.95)
241 (1.29)
239 (3.0)
nda
DMSO
Toluene
nda
nda
nda
a
nd—not determined—insufficient solvent transmittance.

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A. Głuszynska et al. / Tetrahedron Letters 51 (2010) 5415–5418
correlate well with the emission characteristics of ligand 1 in 5%
EtOH which exhibit only a single broad emission band centered
around 550 nm. The position and shape of the long-wavelength
emission band resemble those reported for anthrylvinylpyridinium
derivatives.22 The relative intensities of both fluorescence bands
change with the content of ethanol in solution and can be ascribed
to the extent of dimerization (association) of the ligand. The
reversibility of this association process was confirmed by changing
the solvent mixing order, which gave the same bimodal-emission
spectra at 50% content of EtOH, starting from both 5% EtOH and
pure EtOH solutions of 1.
Preliminary studies on the DNA binding affinity of the ligands
were carried out with calf thymus DNA as a double-stranded
DNA (dsDNA), and 22-mer oligonucleotide (50-AGGGTTAGGGTTA
GGGTTAGG G-30), with a sequence related to human telomere
DNA (G4 DNA), which can form intramolecular G-quadruplex
structures by the folding of a single oligonucleotide molecule.
Small spectral changes were observed in the absorption and emis-
sion spectra of both ligands in the presence of DNA as shown in
Figure 3.
nitrogen) should improve the solubility and DNA binding affinities
of the ligands. Further investigations including new syntheses and
DNA binding studies are in progress.
Acknowledgements
This work was supported by Grant No. N204 004 32/0254 from
the Ministry of Science and Higher Education.
References and notes
1. Li, J.; Li, D.; Han, Y.; Shuang, S.; Dong, Ch. Spectrochim. Acta Part A 2009, 73,
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2008, 90, 1233–1249.
4. De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.; Trentesaux, Ch.; Riou,
J.-F.; Mergny, J.-L. Biochemie 2008, 90, 131–155.
5. Folini, M.; Gandellini, P.; Zaffaroni, N. Biochim. Biophys. Acta 2009, 1792, 309–
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6. Mau, A. W. H.; Sasse, W. H. F.; Creaser, I. I.; Sargeson, A. M. New J. Chem. 1986,
10, 589–592.
7. Juskowiak, B.; Ohba, M.; Sato, M.; Takenaka, S.; Takagi, M.; Kondo, H. Bull. Chem.
Soc. Jpn. 1999, 72, 265–277.
8. Głuszyn´ ska, A.; Rozwadowska, M. D. Tetrahedron: Asymmetry 2000, 11, 2359–
These indicate that the ligands interact with both dsDNA, and
G4 DNA, but spectral effects are rather modest. In the case of ligand
1 small quenching and a blue-shift of fluorescence was observed
(Fig. 3A). The fluorescence spectrum of ligand 2 undergoes oppo-
site changes in the presence of DNA, that is, a red-shift and a small
enhancement effect.
2366.
9. Wen, L.; Li, M.; Schlenoff, J. B. J. Am. Chem. Soc. 1997, 119, 7726–7733.
10. Kagan, F.; Birkenmeyer, R. D.; Strube, R. E. J. Am. Chem. Soc. 1959, 81, 3026–
3031.
11. Compound 4: To a vigorously stirred suspension of 9,10-dibromoanthracene
(1.00 g, 3 mmol) in THF (60 mL) under an argon atmosphere at ꢀ72 °C n-BuLi
(1.6 M solution in hexane, 6.25 mL, 8.35 mmol, 1.4 equiv) was added dropwise.
After 1 h, 4-formylmorpholine (0.72 mL, 7.2 mmol, 1.2 equiv) was added at the
same temperature. The yellow clear mixture was kept at ꢀ35 °C for 12 h and
then quenched with 20% NH4Cl (20 mL) at the same temperature. After
warming to rt, phases were separated and the aqueous one was extracted with
Et2O (3 ꢁ 20 mL). The combined organic extracts were dried, and the solvent
evaporated under reduced pressure to afford a crude product which was
crystallized from CH2Cl2/hexane to give 485 mg (70%) of 9-bromoanthracene-
10-carboxaldehyde (4) as yellow crystals. Mp: 210–213 °C [lit.12 207–208 °C
(C6H6)]. Spectral data were identical with literature data.12
12. Gore, P. H.; Gupta, S. D.; Obaji, G. A. J. Prakt. Chem. 1984, 326, 381–384.
13. Compound 5: To a yellow suspension of 9-bromoanthracene-10-carboxaldehyde
(4) (300 mg, 1 mmol) in DMSO (30 mL) CuCN (895 mg, 10 mmol) was added. The
mixture was heated at reflux with stirring until no more substrate was present
(6 h, TLC). After cooling to rt the copper complex of the product was destroyed
with 25% NH4OH (30 mL). The solid was filtered off, washed with H2O (40 mL),
dried, and was crystallized from CH2Cl2/hexane to give 194 mg (81%) of 9-cyano-
10-anthracenocarboxaldehyde (5) a yellow crystals. Mp: 260–263 °C (lit.12 263–
264 °C). Spectral data were identical with literature data.12
It can be speculated, that the poor solubility of the ligands in
aqueous solution, and the tendency to form associates may hamper
efficient intercalation of the ligands to the DNA samples. The intro-
duction of a positive charge into the ligand structure (quaternary
25000
20000
15000
10000
5000
1
A
dsDNA + 1
G4 DNA + 1
14. Olah, G. A.; Ohannesian, L.; Arvanaghi, M. J. Org. Chem. 1984, 49, 3856–3857.
15. Whitaker, K. E.; Snyder, H. R. J. Org. Chem. 1970, 35, 30–32.
16. Wang, C.; Bryce, M. R.; Batsanov, A. S.; Stanley, C. F.; Beeby, A.; Howard, J. A. K.
J. Chem. Soc., Perkin Trans. 2 1997, 1671–1678.
17. Nakatsuji, S.; Matsuda, K.; Uesugi, Y.; Nakashima, K.; Akiyama, S.; Katzer, G.;
Fabian, W. J. Chem. Soc., Perkin Trans. 2 1991, 861–867.
18. Sun, L.; Görner, H. J. Phys. Chem. 1993, 97, 11186–11193.
40000
2
19. Synthesis of nitriles
7 and 8: To a solution of diethyl[(4-cyanophenyl)-
B
methyl]phosphonate9,10 (278 mg, 1.1 mmol) in DMF (1 mL), freshly prepared
MeONa (0.7 mL, 1.62 mmol, 2.3 M/dm3) was added. The ylide was generated at
room temperature in 10 min under an argon atmosphere. After cooling to 0 °C
aldehyde (1 mmol) in DMF (4 mL) was added. The mixture was stirred at this
temperature or at rt until completion of the reaction (TLC). The resulting
precipitatewas filtered and thefiltrate pouredonto ice. Thesolid was filtered, the
combined solids washed with H2O (20 mL) and hexane (20 mL), and dried to give
the crude product. Compound 7: 3 h, 0 °C. The crude product was crystallized
from CH2Cl2/hexane; yellow crystals (65%), mp: 268–270 °C (hexane–CH2Cl2). IR
(KBr) cmꢀ1: 2224 (C„N), 2211 (C„N), 964 (E CH@CH). 1H NMR (CDCl3) d: 6.96
(d, J = 16.7 Hz, 1H, CH@CH), 7.52–7.62 (m, 2H, ArH), 7.72–7.86 (m, 4H, ArH), 8.00
(d, J = 16.5 Hz, 1H, CH@CH), 8.01–8.04 (m, 2H, ArH), 8.32–8.37 (m, 2H, ArH),
8.46–8.50 (m, 2H, ArH). EIMS: m/z (%): 227 (26), 301 (16), 328 (Mꢀ2, 14), 329
(Mꢀ1, 43), 330 (M+, 100), 331 (M+1, 33), 332 (M+2, 11). HRMS calcd for C24H14N2
330.11569. Found 330.11614.Compound 8: 22 h, rt; the TLC-pure product 8 was
used in the next step without further purification; yellow crystals (yield: 55%),
mp: 186–188 °C. IR (KBr) cmꢀ1: 2220 (C„N), 966 (E CH@CH). 1H NMR (CDCl3) d:
1.45 (t, J = 7.1 Hz, 3H, CH2–CH3), 4.38 (q, J = 7.1 Hz, 2H, CH2–CH3), 7.11 (d,
J = 16.5 Hz, 1H, CH@CH), 7.24–7.29 (m, 1H, ArH), 7.39–7.52 (m, 3H, ArH), 7.55 (d,
J = 16.5 Hz, 1H, CH@CH), 7.58–7.69 (m, 5H, ArH), 8.11–8.14 (m, 1H, ArH). EIMS:
m/z (%): 204 (10), 292 (16), 307 (63), 322 (100). HRMS calcd for C23H18N2
322.14700. Found 322.14591.
dsDNA + 2
G4 DNA + 2
30000
20000
10000
0
B
400
500
600
700
800
Wavelength (nm)
Figure 3. Fluorescence emission spectra of ligand 1 (A): (1.62 ꢁ 10ꢀ5 M, 5% EtOH,
10 mM Tris–HCl pH 7.2, kex = 388 nm) and ligand 2 (B): (1.2 ꢁ 10ꢀ5 M, 5% EtOH,
10 mM Tris–HCl pH 7.2, kex = 350 nm) in the absence (black) and the presence of
dsDNA (1.25 equiv, red) and G4 DNA (1.25 equiv, green).
20. Synthesis of oxazolines 1 and 2. Compound 1: To ( )-1-amino-2-propanol
(1.1 mL, 14 mmol) and anhydrous K2CO3 (81.5 mg) in a mixture of ethylene

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A. Głuszynska et al. / Tetrahedron Letters 51 (2010) 5415–5418
5418
glycol (10.8 mL), and glycerol (5.9 mL) trans-1-[(9-(10-cyanoanthryl)]-2-(4-
ethylene (8) (80.5 mg, 0.25 mmol) was added, and the resulting mixture was
heated at 115 °C with stirring under an argon atmosphere until no more
substrate reacted (ca. 22 h, TLC). After cooling to rt, H2O (10 mL) was added,
and the solid was filtered. The filtrate was extracted with CHCl3 until the
Dragendorff test was negative. The combined organic extracts were dried and
the solvent was evaporated under reduced pressure to afford crude product 2
(64 mg, 67%). This was washed with Et2O (15 mL). The ethereal solution was
concentrated in vacuo to give TLC-pure trans-1-[(9-n-ethyl)-3-carbazole]-2-[4-
(5-methyl-2-oxazoline)phenyl]-ethylene (2) (13 mg, 14%). Mp: 120–122 °C. IR
(KBr) cmꢀ1: 1644 (C@N), 961 (E CH@CH). 1H NMR (CDCl3) d: 1.44 (d, J = 6.3 Hz,
3H, O–CH–CH3), 1.45 (t, J = 7.1 Hz, 3H, CH2–CH3), 3.63 (dd, J = 14.4, 7.4 Hz, 1H,
C@N–CHH), 4.16 (dd, J = 14.4, 9.3 Hz, 1H, C@N–CHH), 4.5 (q, J = 7.1 Hz, 2H,
CH2–CH3), 4.85–4.88 (m, 1H, O–CH–CH3), 7.16 (d, J = 16.2 Hz, 1H, CH@CH),
7.41–7.48 (m, 4H, ArH, 7.46 (d, J = 16.2 Hz, 1H, CH@CH), 7.59 (d, J = 8.2, 3H,
ArH), 7.93 (d, J = 8.5 Hz, 2H, ArH), 8.1 (d, J = 7.41 Hz, 1H, ArH), 8.25 (d,
J = 1.6 Hz, 1H, ArH). ES-MS: (positive mode) m/z 381 [M+H]+, 403 [M+Na]+.
21. Rozwadowska, M. D. Tetrahedron: Asymmetry 1998, 9, 1615–1618.
22. Juskowiak, B.; Chudak, M.; Takagi, N.; Takagi, M. Pol. J. Chem. 2002, 76,
83–93.
cyanophenyl)ethylene (7) (82 mg, 0.35 mmol) was added, and the resulting
mixture was heated at 115 °C with stirring under an argon atmosphere until no
more substrate reacted by TLC (ca. 72 h). After cooling to ambient temperature,
the mixture was poured onto ice and after rt was reached the solid was filtered.
The solid was washed with H2O (20 mL) and hexane (20 mL), and was dried to
give 41 mg (43%) of crude trans-1-[(9-(10-cyanoanthryl)]-2-[4-(5-methyl-2-
oxazoline)phenyl]ethylene (1), which was purified by HPLC using a preparative
column (Inertsil PRER-ODS 100% MeOH, flow 9.9 ml/min, 258 nm. Retention
time = 2.9 min), Mp: 210–215 °C. IR (KBr) cmꢀ1: 2211 (C„N), 1704 (C@N), 975
(E CH@CH). 1H NMR (CDCl3) d: 1.31 (d, 3H, J = 6,3 Hz, CH3), 3.65 (dd, 1H, J = 7.4,
14.6 Hz, C@N–CHH), 4.18 (dd, 1H, J = 9.3, 14.6 Hz, C@N–CHH), 4.89–4.93 (m,
1H, OCH–CH3), 6.96 (d, 1H, J = 16.5 Hz, CH@CH), 7.58–7.62 (m, 4H, ArH), 7.71–
7.74 (m, 2H, ArH), 7.95 (d, 1H, J = 16.5 Hz, CH@CH), 8.03–8.23 (m, 2H, ArH),
8.39–8.45 (m, 2H, ArH), 8.46–8.49 (m, 2H, ArH). EIMS: m/z (%): 388 (M+, 100),
389 (M+1, 37), 390 (M+2, 12). HRMS calcd for C27H20N2O 388.15756. Found
388.15911. Compound 2: To ( )-1-amino-2-propanol (0.175 ml, 2.5 mmol),
and anhydrous K2CO3 (53 mg) in a mixture of ethylene glycol (0.48 ml), and
glycerol
(0.27 ml)
trans-1-[(9-n-ethyl)-3-carbazole]-2-(4-cyanophenyl)-