6-Nitro-Quinazolin?4(3H)?one Exhibits Photodynamic Effects and Photodegrades Human Melanoma Cell Lines. A Study on the Photoreactivity of Simple Quinazolin?4(3H)?ones

Article abstract of DOI:10.1111/php.13376

Photochemo and photodynamic therapies are minimally invasive approaches for the treatment of cancers and powerful weapons for competing bacterial resistance to antibiotics. Synthetic and naturally occurring quinazolinones are considered privileged anticancer and antibacterial agents, with several of them to have emerged as commercially available drugs. In the present study, applying a single-step green microwave irradiation mediated protocol we have synthesized eleven quinazolinon?4(3H)?ones, from cheap readily available anthranilic acids, in very good yields and purity. These products were irradiated in the presence of pBR322 plasmid DNA under UVB, UVA and visible light. Four of the compounds proved to be very effective DNA photocleavers, at low concentrations, being time and concentration dependent as well as pH independent. Participation of reactive oxygen species was related to the substitution of quinazolinone derivatives. 6-Nitro-quinazolinone in combination with UVA irradiation was found to be in vitro photodestructive for three cell lines; glioblastoma (U87MG and T98G) and mainly melanoma (A?375). Thus, certain appropriately substituted quinazolinones may serve as new lead photosensitizers for the development of promising biotechnological applications and as novel photochemo and photodynamic therapeutics.

Full text of DOI:10.1111/php.13376

Photochemistry and Photobiology, 20**, **: *–*
Research Article
6-Nitro-Quinazolinꢀ4(3H)ꢀone Exhibits Photodynamic Effects and
Photodegrades Human Melanoma Cell Lines. A Study on the
Photoreactivity of Simple Quinazolinꢀ4(3H)ꢀones
Anastasios Panagopoulos1†, Thomas Balalas2†, Achilleas Mitrakas3, Vassilios Vrazas4,
Katerina R. Katsani4, Alexandros E. Koumbis2, Michael I. Koukourakis3, Konstantinos E. Litinas2 and
Konstantina C. Fylaktakidou1,2
*
1Laboratory of Organic, Bioorganic and Natural Product Chemistry, Molecular Biology and Genetics Department, Democritus
University of Thrace, Dragana, Alexandroupolis, Greece
2Laboratory of Organic Chemistry, Chemistry Department, Aristotle University of Thessaloniki, Thessaloniki, Greece
3Department of Radiotherapy/Oncology, Democritus University of Thrace/University General Hospital of Alexandroupolis,
Alexandroupolis, Greece
4Laboratory of Biochemistry and Molecular Virology, Molecular Biology and Genetics Department, Democritus University of
Thrace, Dragana, Alexandroupolis, Greece
Received 16 October 2020, accepted 29 December 2020, DOI: 10.1111/php.13376
ABSTRACT
diseases. They usually depend on the excitation of a chemical
compound, a “photosensitizer,” which absorbs light of a specific
wavelength and triggers a series of cascade reactions able to
destroy “on-demand” biological targets, both in a spatial and a
temporal way.
Photochemo and photodynamic therapies are minimally inva-
sive approaches for the treatment of cancers and powerful
weapons for competing bacterial resistance to antibiotics.
Synthetic and naturally occurring quinazolinones are consid-
ered privileged anticancer and antibacterial agents, with sev-
eral of them to have emerged as commercially available
drugs. In the present study, applying a single-step green
microwave irradiation mediated protocol we have synthesized
eleven quinazolinonꢀ4(3H)ꢀones, from cheap readily avail-
able anthranilic acids, in very good yields and purity. These
products were irradiated in the presence of pBR322 plasmid
DNA under UVB, UVA and visible light. Four of the com-
pounds proved to be very effective DNA photocleavers, at
low concentrations, being time and concentration dependent
as well as pH independent. Participation of reactive oxygen
species was related to the substitution of quinazolinone
derivatives. 6-Nitro-quinazolinone in combination with UVA
irradiation was found to be in vitro photodestructive for three
cell lines; glioblastoma (U87MG and T98G) and mainly mela-
noma (Aꢀ375). Thus, certain appropriately substituted
quinazolinones may serve as new lead photosensitizers for
the development of promising biotechnological applications
and as novel photochemo and photodynamic therapeutics.
Moreover, biotechnological phototriggered studies are cur-
rently exploding focusing on approaches which aim to give solu-
tions in critical problems, for example inhibition of teratoma in
stem cell regeneration therapy (9) or improvement of drug deliv-
ery via visible light-triggered intracellular co-delivery and photo-
cleavage of linkers (10). Finally, photosensitizing techniques are
used in the fields of bioimaging and photocatalysis (11,12).
As far as therapeutic approaches are concerned, the efficiency
of the treatments depends not only on the properties of the pho-
tosensitizer but also on the characteristics of the irradiated area
and the wavelength of light irradiation. UVB (290ꢀ315 nm) and
UVA (315ꢀ400 nm), or a combination of them, find extensive
applications mainly, but not exclusively, in dermatology (13–15).
Some characteristic examples are referred to the treatment of
psoriasis (16,17) and vitiligo (18,19), palmoplantar pustulosis
(20), early-stage mycosis fungoides (21), T-cell-mediated dis-
eases (22), pityriasis (23), etc. The visible area of the light spec-
trum is currently used for the photodynamic therapy of multiple
actinic keratoses (24,25), Bowen’s disease and basal cell carci-
noma (26,27), melanotic melanoma (28), acne vulgaris (29), to
mention few.
Photosensitizers are also used for the photoinactivation of
bacteria (PIB). This approach seems to be highly promising, as
one of the last lines of defense for the fight against bacteria
resistance to antibiotics. Development of PIB procedures shows,
at the moment, no obvious risk to reach new dead ends
(30–32), due to the fact that the reactive radical species created
by the photosensitizers attack multiple biological targets on
bacteria.
INTRODUCTION
Photochemo therapy (1,2) and photodynamic therapy (3–8) are
considered minimally invasive approaches for alternative or adju-
vant to classical chemotherapeutic, radiotherapeutic or surgical
therapies of several kinds of cancers and other nonmalignant
*Corresponding author email: kfylakta@chem.auth.gr (Konstantina C. Fylaktaki-
dou)
Several organic compounds, bearing quite diverse structures,
have been identified as DNA photocleavaging agents. These
†The authors contributed equally to this work.
© 2021 American Society for Photobiology
1

2
Anastasios Panagopoulos et al.
Figure 1. Structures of parent quinazolinꢀ4ꢀ(3H)ꢀone and commercially available drugs containing quinozalinone scaffold.
include [1,2,4]ꢀtriazoloꢀ[4,3ꢀa]quinoxalines (33), quinox-
alinꢀ4(5H)ꢀones (34), enediynes (35,36), pyrrolecarboxamide
conjugated 40ꢀbromoꢀacetophenones (37), halophenyl sulfon-
amide derivatives of poly Nꢀmethylimidazoles (38), naphthalim-
ides, (39–41) nitrobenzamido amino-acridine derivatives (42,43),
benzo[b][1,8]naphthyridines (44) and quinolones (45–47),
Nꢀphenylhydroxylamine derivatives (48), Oꢀacyl (49–53),
Oꢀsulphonyl (54,55) and O-carbamoyl (56) oximes, bꢀcarboli-
nes (57,58), pyrazole derivatives (59,60), azido carbonyl com-
pounds (61), etc.
Quinazolinones represent ubiquitous structural scaffolds in
medicinal chemistry which are profoundly considered as “privi-
leged structures.” Not surprisingly, they serve as building blocks
for approximately 150 naturally occurring alkaloids isolated from
a number of families of the plant kingdom, from microorganisms
and animals. Additionally, at least 10 commercially available
drugs used in clinical therapy, such as afloqualone, ispinesib, ral-
titrexed and idelalisib (Fig. 1), feature as pharmacophore the
quinazolinone moiety. As a result, both synthetic approaches and
the observed biological activities (anti-inflammatory, anti-cancer,
anti-microbial, anti-convulsant, anti-hypertensive, anti-diabetic
etc) for quinozalinones have been extensively reviewed (62–67).
Within those studies, some derivatives proved to be DNA bind-
ing and cleaving agents (68–70).
Photostability of drugs is considered important in order to
avoid side effects; nevertheless photosensitivity is the demanded
activity for phototherapeutic applications and protocols. In the
literature, photochemical experiments on quinazolinones afforded
syn-anti photoisomerizations of arylethylene derivatives (71,72)
or Schiff bases (73). A recent photophysical study on some
quinazolinone scaffolds has shown a high photostability (74). As
far as photobiological experiment concerns, a certain derivative
and more specifically, the central nervous acting muscle relaxant
drug “Afloqualone (AFQ),” (75) Fig. 1, caused photosensitive
skin reactions upon administration. The drug was phototoxic to
bacteriophage lambda (76), and it was exhibiting DNA photo-
cleavage activity by photodynamic action (77,78). Our team has
incorporated metal complexation in properly designed quinazoli-
none scaffolds and identified photosensitivity owed to the metal
excitation (79,80). It seems to us that photosensitization of this
important class of compounds represents an active field, not only
in terms of the behavior of quinazolinones toward metal com-
plexation, but also in terms of the photoeffects of the substituents
on this pharmacophore scaffold, which are, to the best of our
knowledge, rarely explored. Thus, due to our continuous interest
in the discovery of organic molecules acting as photosensitizers
and DNA photocleavers (53–56) we have initiated a quina-
zolinone-related project, and investigated photosensitization,

Photochemistry and Photobiology
3
the mixture was degassed with argon for at least 1 h.
1,4ꢀcyclohexadiene (50 lL) was added and the mixture was irradiated
for 2 h at 312 nm (2 9 9 W, 5 cm distance). After completion of the
experiment, the solvent was removed under vacuum and a reaction
sample was analyzed by 1HꢀNMR and LCꢀMS (52,54).
photodisrupting and photodynamic effects of various substituents
on this pharmaceutical frame. In our designing, halogens and
nitro groups, as well as hydroxyl and methyl groups at different
positions of the aromatic framework were incorporated.
All compounds were synthesized using a green microwave-as-
sisted protocol, isolated in very high yields and screened under
UVB irradiation in the presence of supercoiled plasmid DNA
pBR322 in order to investigate their efficacy to photoreact. UV
absorption at higher wavelengths was the factor that allowed the
study with UVA and visible light. For the compounds that
showed the highest DNA photocleavage activity, the photochem-
istry and mechanism of action in the presence of DNA using var-
ious scavengers were studied. Finally, glioblastoma cell lines
U87MG and T98G (ATCCâ HTBꢀ14TM and ATCCâ
CRLꢀ1690TM, respectively) and skin malignant melanoma cell
line Aꢀ375 (ATCCâ CRLꢀ1619TM) were treated with the com-
pound that presented overall the best activity, in vitro.
UV irradiations in MeOH/H2O. Pyrex vials containing samples of 15,
18 and 20 (5 mg) were individually dissolved in MeOH/H2O (9/1)
(3 mL). The mixture was degassed with argon for at least 1 h and then
irradiated for 2 h at 312 nm or 365 nm (2 9 9 W, 5 cm distance). After
completion of the experiment, the solvent was removed under vacuum
and a reaction sample was analyzed by 1HꢀNMR and LCꢀMS. (52,54)
Biological assays. Cell culture experiments. Glioblastoma cell lines
U87MG and T98G (ATCCâ HTBꢀ14TM and ATCCâ CRLꢀ1690TM
,
respectively) and skin malignant melanoma cell line Aꢀ375 (ATCCâ
CRLꢀ1619TM) were used for these experiments. Cells were cultured
under aseptic conditions using DMEM basal medium (31885-023; Gibco)
supplemented with 10% fetal bovine serum (FBꢀ1000/500, Biosera,
UK), 100 units/ml Penicillin and 100 lg mLꢀ1 Streptomycin
(15140ꢀ122, Gibco) and 2mM LꢀGlutamine (25030; Gibco). Both cell
lines were maintained at standard conditions (37°C, 5% CO2) in
humidified atmosphere and were used upon reaching 70–90% confluency.
The proper concentration of 15 was found using titrations with seven
concentrations (0ꢀ1 mM). Fluorescence measurements have been
performed using Resazurin Cell Viability Assay (CA035, Canvax, Spain)
as next described. For the measurement of the cytotoxic effect of
compound 15, 500 cells/well for T98G and U87MG cell lines and 2000
cells/well for A-375 cell line were seeded in a 96-well plate. Cells were
incubated for 30 min with 50 lΜ of 15 and fresh medium was added
before irradiation. Then, cells were irradiated using a radiation source
which emitted a wavelength of 365 nm (UVA). UVA lamp was placed
10 cm over the 96-well plate. Resazurin was added 24 h after irradiation.
A nonirradiated 96-well plate was used as a control, under the same
conditions (81,82).
MATERIALS AND METHODS
All commercially available reagent-grade chemicals and solvents were
used without further purification. pB322 supercoiled plasmid was
purchased from New England Biolabs. NMR spectra were recorded on
an Agilent 500/54 (500 MHz, 125 MHz and 470 MHz for 1H, 13C and
19F, respectively) spectrometer using CDCl3, and/or DMSO ꢀ d6 as
solvent. J values are reported in Hz. Mass spectra were determined using
a Shimadzu LCMSꢀ2010 EV system under electrospray ionization (see
Supporting Information) conditions. All samples containing pBR322
plasmid were irradiated at pH 6.8 with Philips 2 9 9W/01/2P UVꢀB
narrowband lamps at 312 nm, Philips 2 9 9W/10/2P UVꢀA lamps at
365 nm or white light OSRAM DULUX S BLUE. The same lamps were
used for the irradiation in solutions without the plasmid. UV–vis spectra
Resazurin cell viability assay. Incubation with 10% resazurin (7 h)
was followed by fluorescence measurement using 590 nm as emission
wavelength and 530ꢀ560 nm as excitation wavelength in a FLUOstar
Omega microplate reader (BMG LABTECH GmbH, Ortenberg,
Germany). Moreover, culture medium was used as negative control while
5% vitamin C was used as positive control for the full reduction of
resazurin.
were recorded on
a Hitachi U–2001 dual-beam spectrophotometer.
Fluorescence spectra were recorded in solution on a Hitachi F-7000
fluorescence spectrophotometer. MW experiments were performed on a
scientific focused microwave reactor (Biotage Initiator 2.0). All reactions
were monitored on commercially available precoated TLC plates (layer
thickness 0.25 mm) of Kieselgel 60 F254. Calculation of yields was based
on the amount of the directly crystallized product.
RESULTS AND DISCUSSION
General procedure for the synthesis of quinazolinones 12ꢀ22. The
commercially available anthranilic acids 1ꢀ11 (1 mmol) were
individually mixed with triethyl orthoformate (1.3 mmol) and ammonium
acetate (1.3 mmol) in the presence of 95% EtOH (0.5 mL). The mixture
was irradiated for 20 min at 110 °C and then allowed to cool to room
temperature. 95% EtOH (0.5 mL) was added and the resulting
suspension was filtered under reduced pressure. The obtained product
was dried under vacuum and was used without any further purification.
(See Supporting Information for references, data analysis and crude
spectra).
DNA photocleavage experiments. Cleavage of supercoiled circular
pB322 DNA by Quinazolinones 12ꢀ22. Compounds 12ꢀ22 were
individually incubated with plasmid DNA at the desired concentration, in
pyrex vials (10 mm diameter) and were irradiated with UVꢀVisible light
(312 nmꢀ18 W, or 365 nmꢀ18 W, or visible light 400ꢀ700nmꢀ18W)
under aerobic conditions at room temperature for 30 min and in 15 cm
distance (312 nm), or 2 h and in 10 cm distance (365 nm), or 2 h and in
10 cm distance (visible light). Conditions of the photobiological reaction
and gel electrophoresis, quantification of DNA-cleaving activity and
calculation of ss% and ds% damage protocols have been described
previously (53). All experiments were performed at least three times.
Photochemical experiments. UV irradiations in DMSOꢀd6. Pyrex
vials containing samples of 15 and 18 were individually dissolved in
DMSOꢀd6 and the solutions were irradiated at 312 nm for 30, 60 and
120 min under air. Additionally, another series of same solutions were
extensively degassed with argon, for at least one hour and then irradiated
for the same time intervals as the solutions before. Finally, 1HꢀNMR
spectroscopy was used to analyze all samples and the changes were
comparatively studied.
Synthetic procedure
Commercially available anthranilic acids 1ꢀ11, triethyl orthofor-
mate and ammonium acetate were added in EtOH. The reaction
mixture for each starting anthranilic acid was subjected to micro-
wave irradiation for 20 min at 110°C, resulting in very good
yields a single product (12ꢀ22, Scheme 1), which was com-
pletely dissolved in the hot reaction mixture. Upon cooling to
room temperature, simple filtration of the precipitated solid and
washing with cold EtOH gave the desired products which needed
no further purification since the byproducts of the reactions
(EtOH and AcOH) were completely removed.
The microwave-assisted reaction of anthranilic acid 1, tri-
ethyl orthoformate and ammonium acetate for the synthesis of
12 was previously performed, solvent-free, in a domestic micro-
wave oven giving the product in excellent yield and purity
(83). The focus of that approach was on the preparation of five
alkyl or phenyl quinazolinone derivatives altered on position 2
of the scaffold. Within this manuscript, the scope of the reac-
tion has been extended, using a variety of additional 10 anthra-
nilic acids, substituted on the aromatic ring with electron
donating or withdrawing groups, using a scientific microwave
reactor.
The use of a minimum amount of ethanol as solvent, demon-
strates the green dimension of this approach, since the same
UV irradiations in Benzene. Pyrex vials containing samples of 15, 18
and 20 (5 mg) were individually dissolved in dry benzene (2 mL) and

4
Anastasios Panagopoulos et al.
Scheme 1. Synthesis of quinazolinꢀ4ꢀ(3H)ꢀones 12ꢀ22. Reagents and conditions: anthranilic acid 1ꢀ11, triethyl orthoformate, ammonium acetate,
20 min, 110°C, EtOH.
solvent is simultaneously used for the crystallization of the
quinazolinone products, providing pure crystals of compounds
12–22 upon cooling of the reaction mixture. Their 1H and
13CꢀNMR spectra (see Supporting Information) are in full
accordance with those reported in the literature. (84,85) By per-
forming this green and efficient synthesis of the simplest quina-
zolinones with diverse substitution pattern on the aromatic ring,
we set up the basis for their photochemical and photobiological
study.
The designing of the experiments included the preliminary
screening of compounds 12–22 in DMSO solutions (500 ll) up
to 10% in a Tris buffer solution (25 lM, pH = 6.8) containing
the supercoiled circular pBR322 DNA (Form I). Their screening
involved UVB (312 nm) or UVA (365 nm) irradiation for
30 min and 2 h, respectively, at room temperature under aerobic
conditions. Compounds 15, 18 and 19 showed considerable
activity, whereas quinazolinones 17 and 22 showed low to mod-
erate activity at this concentration at 312 nm (Fig. 2, Lanes 6, 9,
10, 8 and 13, respectively).
As it is observed in Fig. 2, 6ꢀnitroꢀquinazolinone 15
degraded completely plasmid DNA, designating this compound
as a very efficient one. On the contrary, 7ꢀnitroꢀquinazolinone
20 showed no activity (Lanes 6 and 11, respectively). Neverthe-
less, the latter compound cleaved DNA at higher concentration
(1 mM) giving the nicked form (100%), probably due to its
lower UV absorption in this irradiation area. The rest of the inac-
tive compounds showed no improvement under the same condi-
tions (data not shown). It is worth mentioning that the position
of nitro group in photosensitizers seems to be crucial for the out-
come of the photocleavage, as it was reported for various deriva-
tives. However, due to the diversity of those substrates
(naphthalimides, nitrobenzamidoacridines and N-methyl-pyrrole-
carboxamides) a prediction of the role of the position may lead
DNA photocleavage experiments
One of the most feasible experiments for the discovery of photo-
sensitizers with biological interest seems to be the identification
of DNA “photocleavers,” (86,87) which via various mechanistic
pathways may lead to single-stranded (ss) DNA damage, repair-
able by enzymatic processes and/or double-stranded (ds) DNA
photocleavage (88,89). The requirement for the activity of the
photosensitizer is, besides the ability to be excited at a specific
light wavelength, the affinity to DNA (53,86), which maybe
expressed via intermolecular forces or intercalation. The fact that
these forces could be applicable to other biological targets ren-
ders DNA photocleavage identification a multi-informative pro-
cedure, useful both in chemistry and in biology.
Figure 2. Efficiency of quinazolinones 12ꢀ22 on DNA photocleavage. Gel electrophoresis image (GEI): Lanes 1 and 2: Control plasmid pBR322 and
control pBR322 irradiated; Lanes 3–13: pBR322 + one of quinazolinones (12 or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, respec-
tively) + UV irradiation; Lanes 14 and 15: pBR322 + DMSO (10% and 20%, respectively) + UV irradiation. Calculation % of the formation of nicked
(Form II) and linear (Form III) pBR322 is given at the bottom of the image (53).

Photochemistry and Photobiology
5
Figure 3. Concentration dependence DNA photocleavage of quinazolinones 15 and 20. GEI: Left: 312 nm; Lanes 1–4: pBR322 + 15 (1 or 10, or 50,
or 100 ll, respectively) + UV irradiation; Lane 5: pBR322 + 15 (500 ll) DARK; Center: 365 nm; Lanes 6–9: pBR322 + 15 (1 or 10, or 25, or 50
ll, respectively) + UV irradiation; Right: 365 nm; Lanes 10–14: pBR322 + 20 (1 or 10, or 25, or 50, or 100 lΜ, respectively) + UV irradiation. For
calculations see comment in Fig. 2.
to uncertain conclusions and, thus, for the moment this seems to
be a case study for each class of compounds. (41,42,60) As far
as the halogenated compounds concerns, we observed that 6ꢀflu-
oro quinazolinone 16 has a negligible activity, which was
slightly improved when fluorine was replaced by chlorine (17)
and this trend reached the top of activity almost equally for
6ꢀbromo (18) and 6ꢀiodoꢀquinozalinone (19), Fig. 2 (Lanes 7,
8, 9 and 10, respectively).
It is interesting to note that the activity was almost lost when
chlorine was found at position 7 of the quinazolinone aromatic
ring instead of position 6 (Lanes 8 and 12, respectively), simi-
larly to the results observed for compounds 15 and 20. Addition-
ally, the introduction of a second atom of bromine led to the loss
of almost half of the activity as compared to the activity of com-
pound 18 (Lanes 13 and 9, respectively).
Irradiation at 365 nm was applied to compounds 13, 15, 16
and 20 since they absorb UV light even slightly at this wave-
length (Supporting Information, Figure S3), with derivatives 15
and 20 to absorb notably high, due to their chromophore, the
nitro group. The latter compounds degraded the pBR322 plasmid
at 500 lΜ to such an extent that ethidium bromide did not
detect any plasmid fragment. Thus, the effective concentration
for photocleavage of the 50% of DNA was tested for derivative
15 at 312 and 365 nm and for 20 at 365 nm, Fig. 3.
For derivative 15 this concentration was between 10 and
50 ll at 312 nm (Fig. 3, Lanes 2 and 3) and at ca. 10 ll at
365 nm (Lane 7). Compound 20 destroyed 50% of the plasmid
DNA at ca. 50 ll (Lane 13). Quinazolinone 15 was found to be
inactive in dark, without UV irradiation (Lane 5). Additionally,
none of the tested compounds showed any activity toward DNA
in the absence of UV irradiation. The effective concentration for
photocleavage of the 50% of DNA caused by compound 18 was
100 ll (data not shown), which is rather high and may be attrib-
uted to the different mechanism of action, compared to com-
pound 15 at 312 nm (vide infra).
Compounds showing the optimum DNA photocleavage at
UVA irradiation and absorption behind 365 nm were also exam-
ined under visible light (Fig. 4). 6-Νitro-quinazolinone (15)
exhibited activity at 250 ll (Lane 2). This was gradually and
linearly progressed degrading completely DNA at 500 lΜ and
behind. There was no activity in dark at 1000 ll (Lane 6). On
the contrary, although compound 20 was relatively active at
365 nm, the same linearity between concentration and activity
was not observed (Fig. 4, Lanes 8-10), probably due to its gener-
ally lower photoreactivity compared to that of 15.
We next examined the mechanism of action and the depen-
dence on pH of the most active compounds 15, 18 and 20 under
irradiation at various wavelengths. The activity of 15 against
DNA did not show any change in a wide range of pH (50 ll
concentration, pH 5 to 10, see Supporting Information, Fig-
ure S4.1). Concerning the mechanism of action, it seems that
oxygen plays an important role for the nitro derivatives, whereas
Br compound 18 showed certain differences. As it is observed in
Figs. 5 and 6, compound 15 follows the same mode of action
under all tested irradiation wavelengths (312 nm, 365 nm and
visible light). Under argon, the DNA photocleavage was notably
decreased [Fig. 5, Lanes 3 (312 nm) and 11 (365 nm), and
Fig. 6, Lane 2 (visible light)]. The implication of radicals is
obvious, since the activity was again diminished in the presence
of scavengers, such as Lꢀcysteine and potassium iodide. We
observed no activity of 15 in the presence of the singlet oxygen
scavenger NaN3, an indication that singlet oxygen is involved,
although there was no change in the presence of D2O [NaN3:
Fig. 5, Lanes 6 (312 nm) and 14 (365 nm), and Fig. 6, Lane 5
Figure 4. Concentration dependence DNA photocleavage of quinazolinones 15 and 20 under visible light. GEI: Left and Center: Lanes 1–5:
pBR322 + 15 (50 or 250, or 500, or 600, or 800 ll, respectively) + UV irradiation; Lane 6: pBR322 + 15 (1000 ll) DARK; Right: Lane 7: Control
plasmid pBR322 irradiated; Lanes 8–10: pBR322 + 20 (1000 or 750, or 500 ll, respectively) + UV irradiation. For calculations, see comment in
Fig. 2.

6
Anastasios Panagopoulos et al.
Figure 5. Mechanistic studies for quinazolinone 15. GEI: Left image: 312 nm (50 lΜ). Right image: 365 nm (25 ll). Lanes 1 and 9 are control plas-
mid DNA + UV irradiation; Lanes 2 and 10: pBR322 + 15 + UV; Lanes 3 and 11: pBR322 + 15 + UV + Argon; Lanes 4 and 12: pBR322 + 15 +
UV + Lꢀcysteine (1500 ll); Lanes 5 and 13: pBR322 + 15 + UV + KI (250 ll); Lanes 6 and 14: pBR322 + 15 + UV + NaN3 (20 ml); Lanes 7 and
15: pBR322 + 15 + UV + D2O; Lanes 8 and 16: pBR322 + 15 + UV + DMSO (20 ll). For calculations see comment in Fig. 2.
Figure 6. Mechanistic studies for quinazolinones 15 and 18. GEI: Left image: Visible light (400ꢀ750 nm), (15, 600 ll). Right image: 312 nm (18,
500 ll). Lanes 1 and 8: pBR322 + 15 or 18 + light; Lanes 2 and 9: pBR322 + 15 or 18 + light + argon; Lanes 3 and 10: pBR322 + 15 or 18 + light
+ Lꢀcysteine (1500 lΜ); Lanes 4 and 11: pBR322 + 15 or 18 + light + KI (250 ll); Lanes 5 and 12: pBR322 + 15 or 18 + light + NaN3 (20 ml);
Lanes 6 and 13: pBR322 + 15 or 18 + light + D2O; Lanes 7 and 14: pBR322 + 15 or 18 + light + DMSO (20 ll). For calculations see comment in
Fig. 2.
(visible light); D2O: Fig. 5, Lanes 7 (312 nm) and 15 (365 nm),
and Fig. 6, Lane 6 (visible light)]. The presence of hydroxyl rad-
icals, OH, is also evidenced by the reduction of the activity in
the presence of 20% DMSO [Fig. 5, Lanes 8 (312 nm) and 16
(365 nm), and Fig. 6, Lane 7 (visible light)]. The same trend
was observed for compound 20 at 365 nm (Supporting Informa-
tion, Figure S4.2).
Various nitro-aromatic derivatives were measured to have rel-
atively large triplet yields and rapid radiationless decay via
energy transfer (90,91). The formation of singlet molecular oxy-
gen was found to occur upon irradiation of 7-nitro-1,4-benzodi-
azepines (92), whereas environmental toxicity of nitro-
polyaromatic hydrocarbons has been attributed to reactive oxy-
gen species and oxidative stress caused under sunlight (93).
A completely different behavior was observed for bromo
derivative 18. Its activity remained almost the same under argon
and in the presence of radical scavengers. Some implication of
singlet oxygen was also found (Fig. 6, Right image). Thus, it
seems that the activity was most probably due to both homolysis
of the CꢀBr bond as well as singlet oxygen formation pathway
(37,38). Due to the similar nature and photocleavage at 312 nm
between the bromo and the iodo quinazolinone derivatives (18
and 19, respectively, Fig. 2) we assume the same mode of action
for compound 19.
macromolecule. Nevertheless, some mechanistic aspects may,
indeed, be clarified. The following photochemical experiments
aimed to observe a possible reactivity of the NO2 groups or homol-
ysis of the CꢀBr bond. Any information was validated in order to
further verify the observed photobiological results (Figs. 5 and 6).
In this context, compounds 15 and 18 were irradiated at
312 nm for 30, 60 and 120 min under air and under Argon (after
careful degassing of the solutions) in a deuterated solvent
(DMSOꢀd6). 1HꢀNMR spectroscopy was used to check all
samples and the changes were compared to nonirradiated sam-
ples. As far as compound 15 concerns, we could observe no
changes in our samples by 1HꢀNMR, neither in the presence
nor in its absence (see Supporting Information, Figure S5.1). For
compound 18, under both conditions, we could observe in the
spectrum the appearance of some new low-intensity peaks which
correspond probably to compound 12, meaning that there was a
tendency of quinazolinone 18 to lose bromine radical. However,
this transformation occurs very slowly, under these conditions
(see Supporting Information, Figure S5.2). Therefore, it seems
that irradiation in DMSO solutions most probably does not cause
any change to the compounds. This finding is in accordance with
the blank experiments shown in Fig. 2 and verifies that DMSO
does not affect, individually, either the plasmid DNA at the con-
centrations used or the compounds at any concentration, upon
irradiation. However, the experiments shown below, in environ-
ments able to provide hydrogen species indicated a different
behavior profile.
.
Photochemical experiments
Compounds which photocleave DNA should be able to form a
DNA-compound type of “complex” via various intermolecular
forces (53). The affinity to DNA obviously changes their photo-
chemistry as compared to that in solution at the absence of the
The tendency of 1,4ꢀcyclohexadiene to supply hydrogen
atoms (conversion to benzene) could be used to determine
whether our compounds may abstract hydrogen atoms. If this is
proved true, then the compounds should have the possibility to

Photochemistry and Photobiology
7
react similarly, with DNA. Compounds 15, 18 and 20 were left
to react with 1,4ꢀcyclohexadiene in dry benzene under argon,
upon irradiation at 312 nm for 2 h. As expected, we observed
improvement of therapeutic protocols as well as the understand-
ing of their biochemical pathways is of our high interest. (94–
99) Thus, the possible photocytotoxic effect of compound 15
against glioblastoma T98G and U87MG as well as melanoma
Aꢀ375 cell lines has been evaluated.
1
no change in the HꢀNMR spectra of the crude reaction mixture
of compounds 15 and 20, and LCꢀMS analysis showed peaks
of the starting material and only traces of other compounds (Sup-
porting Information, Figures S6 and S7). However, the
1HꢀNMR spectrum of the reaction mixture of bromo derivative
18 had extra peaks, which correspond to the CꢀBr homolysis
derivative 12 (Supporting Information, Figure S8).
The Resazurin Cell Viability Assay (CA035, Canvax, Spain)
was used for cytotoxicity studies. This is a reliable, simple, non-
toxic and safe method for cell viability based on the metabolic
activity of cells to reduce resazurin to resorufin. In brief, cells
seeded on well plates were incubated with 50 ll of 15 and then
irradiated at 365 nm using a UVA lamp for 1 and 2 h. Control
cells were also irradiated with UVA for 2 h.
The extent of conversion of compound 18 was better notice-
able when experiments were performed changing the solvent to
MeOH/H2O (9/1) under irradiation at 312 nm for 2 h. By ana-
lyzing with LCꢀMS we observed the appearance of two bands
of ions at 4.5ꢀ5.4 min which correspond to unreacted compound
18 and the newly formed derivative 12. The same results were
In glioblastoma cell lines it was observed that 15 did not
affect cells’ viability in dark (Fig. 7, 2nd and 5th column from
left to right). Its cytotoxic effect appeared after irradiation and it
is time dependent since it is increased, significantly after 2 h of
UVA irradiation. Nevertheless, cytotoxicity was higher for T98G
rather than U87MG. UVA irradiation did not affect cells’ viabil-
ity (data not shown).
Similarly, 15 was not cytotoxic for A375 cells without
UVA irradiation (Fig. 8). However, A375 cell population was
reduced dramatically when followed incubation with 15 and
UVA irradiation. Control A375 cell lines population has been
reduced by 60% when simply irradiated with UVA, while
only 1ꢀ5% of cells survived when incubation with 15 was
combined with UVA irradiation. Cytotoxic effect of 15 was
weaker when cells were irradiated after removal of 15. This is
an indication that, during the experimental incubation time,
compound 15 may enter cells and cause some damage, upon
irradiation. Nevertheless, it seems that the remaining of the
compound in the cell growth medium is able to cause further
damage, probably on cell’s membranes or other organelles,
1
observed in the HꢀNMR spectrum of the crude irradiated mix-
ture. The ratio of compounds 12 and 18 was 1/1 (ratio of the
corresponding hydrogen peaks, see Supporting Information, Fig-
ure S8). In general, homolysis of the Cꢀhalogen bond was
found to be the driving force and the cause of DNA damage in
other halogenated DNA photocleavers, (37,38) as well. No such
homolysis was realized under irradiation at 365 nm, due to the
lack of UV absorbance of the compound. Both nitro derivatives
1
15 and 20, showed no change by HꢀNMR and LCꢀMS analy-
sis under either UVB or UVA irradiation when subjected to simi-
lar experimental conditions.
Biological assays
Cell culture experiments. Glioblastoma and melanoma are
aggressive lethal human tumors for which the search for the
Figure 7. Cytotoxic effect of compound 15 against T98G and U87MG cell lines.

8
Anastasios Panagopoulos et al.
Figure 8. Cytotoxic effect of compound 15 against A375 melanoma cell line.
making, thus, compound 15 a promising tool for further pho-
todamaging studies.
7ꢀnitro derivative 20 showed considerable activity at UVA and
visible light. Formation of reactive oxygen species was found to
be responsible for their activity. Nitro derivative 15, which
exhibited the best profile, was further tested in cells. It was
found to be a very efficient in vitro photosensitizer for skin
malignant melanoma cell line Aꢀ375, whereas its activity
against two glioblastoma cell lines (U87MG and T98) was mod-
erate.
Conclusively, it seems, that certain substituted quinazolinones,
bearing a Br, I or NO2 atom/group at position 6, may serve as
new lead photosensitizers for the development of promising
biotechnological applications and as novel photochemo and pho-
todynamic therapeutics. Additionally, the designing of novel
pharmaceutics based on certain quinazolinones should take into
consideration their possible photoreactivities.
CONCLUSION
Eleven quinazolinones were synthesized in a one-step procedure
from cheap readily available anthranilic acids, in very good
yields and purity, using a green microwave irradiation mediated
approach. Screening of these compounds in the presence of
pBR322 plasmid DNA under UVB, UVA and visible light
revealed four very effective DNA photocleavers, being time and
concentration dependent and pH independent. Oxygen was par-
ticipating on the DNA photocleavage; however, it was crucial
for the nitro derivatives, indicating a possible photodynamic
behavior.
Parent quinazolinone 12, as well as 6ꢀMe (13) and 6ꢀOH
derivatives (14) were not active. On the contrary, 6ꢀbromo
derivative 18 (and most probably iodo derivative 19) exhibited
its reactivity upon UVB irradiation via Cꢀhalogen bond homoly-
sis as well as via singlet oxygen. Fluoro and chloro derivatives
showed limited activity. Comparing the 6ꢀor 7ꢀchloro quina-
zolinones it becomes evident that substitution at position 6 is
important for photoreactivity. The same was concluded for
6ꢀand 7ꢀnitro derivatives 15 and 20, respectively. Addition of
a second halogen atom, as in the case of 6,8ꢀdiꢀbromo
Acknowledgements—Part of this work was realized using the facilities of
Hellenic Small Molecules Center (Aristotle University of Thessaloniki),
partner of OPENSCREENꢀGR (MIS 5002691), which is implemented
under the Action “Reinforcement of the Research and Innovation
Infrastructure,” funded by the Operational Program “Competitiveness,
Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by
Greece and the European Union (European Regional Development
Fund).
derivative 22 resulted in
a considerable reduction of the
SUPPORTING INFORMATION
photosensitivity.
6ꢀNitro derivative 15 was highly active at UVB, UVA (at
Additional supporting information may be found online in the
Supporting Information section at the end of the article:
concentrations even below 10 lΜ) and visible light, whereas

Photochemistry and Photobiology
9
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Data S1. Data for quinazolinones 12ꢀ22.
Data S2. Copies of NMR spectra of quinazolinones 12ꢀ22.
Data S3. UVꢀVis spectra of quinazolinones 12ꢀ22.
Data S4. Gel electrophoresis studies: Figure S4.1. Compound
15, study under various pH, Figure S4.2. Compound 20, mecha-
nistic study at 365 nm.
€
15. Herzinger, T., M. Berneburg, K. Ghoreschi, H. Gollnick, E. Holzle,
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Kochanek, T. Schwarz, J. Simon, A. Tanew and M. Weichenthal
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Data S5. H NMR spectra of compounds 15 and 18 after irra-
diation in DMSOꢀd6 at 312 nm: Figure S5.1. Compound 15,
Figure S5.2. Compound 18.
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Data S6. H NMR spectra and LC-MS analysis of compound
15 after irradiation a) in MeOH/H2O at 365 nm, b) in MeOH/
H2O at 312 nm, and c) in benzene with 1,4-cyclohexadiene.
1
Data S7. H NMR spectra and LC-MS analysis of compound
20 after irradiation a) in MeOH/H2O at 365 nm, b) in MeOH/
H2O at 312 nm, and c) in benzene with 1,4-cyclohexadiene.
1
Data S8. H NMR spectra and LC-MS analysis of compound
18 after irradiation a) in MeOH/H2O at 365 nm, b) in MeOH/
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Data S9. Fluorescence spectra of quinazolinones 12ꢀ22.
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