2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P90 EPR STuDy ON PhOTOINDuCED PROCESSES
OF NOVEL QuINOLONE DERIVATIVES
ZUZAnA VRECKOVá, VLASTA BREZOVá, MAROŠ
BELLA, VIKTOR MILATA and SOňA JAnTOVá
Institute of Physical Chemistry and Chemical Physics, Faculty
of Chemical and Food Technology, Slovak University of
Technology in Bratislava, Radlinského 9, 812 37 Bratislava,
Slovak Republic,
zuzana.vreckova@stuba.sk
Introduction
4-Oxo-1,4-dihydroquinoline derivatives (4-quinolones)
represent one of the largest classes of antimicrobial agents
used nowadays in the medical care 1 . Specific members of
this drug family display also high activity against eukaryotic
type II topoisomerases, as well as against cultured mammalian
cells 2 . These antineoplastic quinolones represent a prospective
source of new anticancer agents. The presence of
extended π-electron system of quinolone derivatives results
in their photosensitive properties; therefore UVA irradiation
can induce their photosensitive reactions with phototoxic
responses. Previously we demonstrated that excitation of nitrogen
heterocycle molecules by UVA radiation may significantly
enhance their biological activities 3 .
novel 7-substituted 6-oxo-6,9-dihydro[1,2,5]selenadiaz
o-lo[3,4-h]quinoline derivatives were synthesized as potential
anticancer and antimicrobial agents and their ability to
produce Reactive Oxygen Species (ROS) upon irradiation
was tested using Electron Paramagnetic Resonance (EPR)
spectroscopy. Additionally, their cytotoxic/phototoxic effects
on human leukemia cells HL60 were characterized.
Experimental
The synthesis of 7-substituted 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinoline
derivatives was performed
according to the reaction pathways published in ref. 4 .
Table I summarizes the structure, substitution and abbreviation
of the synthesized selenadiazoloquinolones.
E P R P h o t o c h e m i c a l E x p e r i m e n t s
EPR measurements at the X-band were performed with
an EMX EPR spectrometer (Bruker, Germany) using a TE 102
(ER 4102ST) resonator. Samples were irradiated at 295 K
directly in the EPR spectrometer cavity using HPA 400/30S
lamp (400 W, l max = 365 nm, Philips, UVA irradiance
3 mW cm –2 ). A Pyrex glass filter was applied to eliminate
the radiation wavelengths below 300 nm. The experimental
EPR spectra acquisition and simulation was carried out using
WIN EPR and SimFonia programs (Bruker).
The photoinduced generation of free radicals was monitored
by spin trapping technique with 5,5-dimethyl-1-pyrroline
n-oxide (DMPO) and 5-(diisopropoxyphosphoryl)-5-methyl-
1-pyrroline n-oxide (DIPPMPO) as the spin traps. The selective
oxidation of 4-hydroxy-2,2,6,6-tetramethylpiperidine
(TMP) via singlet oxygen to the paramagnetic nitroxyl radi-
s521
cal 4-hydroxy-2,2,6,6-tetramethylpiperidine n-oxyl (Tempol)
was applied for 1 O 2 detection by EPR spectroscopy. The
aerated solutions of 7-substituted [1,2,5]selenadiazolo[3,4h]quinolo-nes
in dimethylsulphoxide (DMSO) mixed immediately
before EPR experiments, were transferred into a flat
cell (WG-808-Q, Wilmad) suitable for TE 102 cavity, and the
EPR spectra were monitored in situ.
UV/visible spectra were recorded in DMSO by means of
a Shimadzu UV-3600 spectrophotometer.
Table I
Structure, substitution and abbreviation of investigated 7substituted6-oxo-6,9-dihydro[1,2,5]-selenadia-zolo[3,4h]quinoline
derivatives
Structure 7-Substitution Abbreviation
H 7-H-SeQ
COOC 2 H 5 7-COOEt-SeQ
COOCH 3 7-COOMe-SeQ
COOH 7-COOH-SeQ
COCH 3 7-Ac-SeQ
Cn 7-Cn-SeQ
C y t o t o x i c / P h o t o t o x i c E f f e c t
The murine leukemia cell line HL60 (obtained from
American Type Culture Collection, Rockville, MD, USA)
was used. These cells were grown in RPMI medium in 5 %
CO 2 at 37 °C under conditions specified in ref. 4 . A starting
inoculum 2.6 × 10 5 HL60 cells ml –1 in the exponential phase
of growth was used. 5 ml of the suspension were added
into Petri dishes (diameter 60 mm), then 20 μl of derivative
7-Ac-SeQ at various concentrations were added to the cells.
One part of the dishes was irradiated with HPA 400/30S lamp
upon UVA dose of 0.4 J cm –2 . After 24, 48 and 72 h cultivation,
the number of cells per culture dishes was counted in a
Bürker chamber and viability of treated and control irradiated/non
irradiated cells were determined by 0.4 % trypan
blue staining.
Results
7-Substituted 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[
3,4-h]quinoline derivatives absorb UV radiation with three
absorption maxima about 400, 340 and 300 nm, which are
only slightly influenced by the substituents properties, as is
shown in Fig. 1. The presence of acetyl substituent in 7-Ac-
SeQ derivative caused bathochromic shift of the low-energy
band to 410 nm with a shoulder at 485 nm.
Upon photoexcitation, all 7-substituted 6-oxo-6,9-dihy
dro[1,2,5]selenadiazolo[3,4-h]quinoline derivatives demonstrated
the ability to generate super-oxide anion radicals trapped
as • DMPO-O 2 – or • DIPPMPO-O2 – spin adducts.
Fig. 2.a shows the time-course of 11 individual EPR
spectra monitored upon continuous UVA irradiation of 7-
COOEt-SeQ, indicating the efficient generation of twelveline
EPR signal with spin Hamiltonian parameters corresponding
to • DMPO-O 2 – (an = 1.275 mT; a H β = 1.032 mT;