Evaluation of Perylenediimide Derivatives for Potential Therapeutic Benefits on Cancer Chemotherapy

Article abstract of DOI:10.1111/cbdd.12004

Perylene derivatives, known to have potential therapeutic benefits on particular cancer types as photosensitizers, may also function as small-molecule inhibitors with promising therapeutic value for diverse diseases. This recently recognized biological activity was attributed to their capacity to modulate the function of various enzymes as biological targets in vitro. Although the inhibitory activity on glutathione transferase and Src tyrosine kinase is important in determining the anticancer potential of compounds for target-specific drug design and development, to date, there are no successful inhibitors of this kind. Moreover, there are only a few studies about the effects of perylene derivatives on glutathione transferase and various kinases. In this study, four novel perylene compounds, N,N′-disubstituted perylenediimides and their 1,7-dibromo derivatives, were synthesized and evaluated for their biological activities. Here, among the compounds analyzed, one of them was identified with strong glutathione transferase inhibition and two with dual activity for both glutathione transferase and c-src inhibition. These results revealed that perylene derivatives may be employed as potential chemosensitizers to prevent chemotherapy-dependent drug resistance and identified as prospective anticancer agents with dual activity on both glutathione transferase and c-src enzymes.

Full text of DOI:10.1111/cbdd.12004

ª 2012 John Wiley & Sons A/S
Chem Biol Drug Des 2012
doi: 10.1111/cbdd.12004
Research Article
Evaluation of Perylenediimide Derivatives for
Potential Therapeutic Benefits on Cancer
Chemotherapy
Taner Keskin1, Belgin S. Isgor2, Yasemin
G. Isgor2 and Funda Yukruk1,*
cells, organic light-emitting diodes, semiconductors and organic
thin film transistors, as well as in chemical oxidations as photo-
sensitizers (1–3). The most commonly analyzed perylene derivatives
were from hypericin (1,3,4,6,8,13-hexahydroxy-10,11-dimethylphen-
1Department of Chemistry, Faculty of Art and Sciences, Balikesir
University, Cagis, Balıkesir 10145, Turkey
anthro[1,10,9,8-opqra]perylene-7,14-dione), which is the most
potent, naturally occurring pro-oxidant that facilitate, or induce,
the apoptosis and necrosis in a wide spectrum of cancer cell
types (4). Moreover, these compounds have shown some antiviral,
antidepressant, and antibacterial activities, and still in use as part
of natural food supplements available in the markets (4–6). The
current use of hypericin and other perylene derivatives in biomedi-
cal applications is usually based on their capacity to produce free
radicals or reactive oxygen species (ROS) upon radiation, so-called
photodynamic therapy (7). Here, the nontoxic compound, adminis-
tered systemically, locally, or topically, acts as photosensitizer and
during the tissue irradiation becomes active and generate ROS.
Eventually, the increased ROS levels cause cytotoxicity on tumor
cells leading to death and tissue destruction (8).The toxicity of
non-radiated photosensitizer is a critical issue for these applica-
tions, because the administered compound directly interacts with
metabolic detoxification system enzymes, and upon irradiation, the
resulting product may become less effective or more toxic to the
organism. As a defense mechanism, the detoxification system
enzymes are evolved and can be induced to protect the organism
against toxic effects of chemicals that may be harmful to their
survival (9). Among these enzymes, mammalian glutathione trans-
ferase (GST, EC 2.5.1.18) is one of the most important detoxifica-
tion enzymes that catalyze the nucleophilic addition of glutathione
to diverse electrophilic molecules (10) and solubilize them to facili-
tate the transport of toxic substances from cells. On the other
hand, GSTs have emerged as promising therapeutic targets
because the specific isozymes are either overexpressed, or overac-
tivated, in a wide variety of diseases, including cancer. Further-
more, current evidence showed the role of GSTs in signal
transduction events is fundamental for cellular functioning (10–12)
through the protein tyrosine kinase (PTK)–mediated GST activation
and signaling from cell membrane to nucleus (13–17).Signal trans-
duction is under tight control, provided by signaling components,
such as kinases, and also by detoxification system enzymes, which
are mostly the GSTs. For therapeutic purposes, the use of small
molecules interfering with signaling components was shown to
induce drug resistance as a result of GST-mediated detoxification
reactions or signaling events. Therefore, in this context, a series
of perylenediimides has been evaluated for their ability to bind
DNA and their potential to induce cytotoxic effects on multiple
2Department of Chemical Engineering and Applied Chemistry,
Faculty of Engineering, Atilim University, Golbasi, Ankara 06836,
Turkey
*Corresponding author: FundaYukruk, fundayukruk@gmail.com
Perylene derivatives, known to have potential
therapeutic benefits on particular cancer types as
photosensitizers, may also function as small-mole-
cule inhibitors with promising therapeutic value
for diverse diseases. This recently recognized bio-
logical activity was attributed to their capacity to
modulate the function of various enzymes as bio-
logical targets in vitro. Although the inhibitory
activity on glutathione transferase and Src tyro-
sine kinase is important in determining the anti-
cancer potential of compounds for target-specific
drug design and development, to date, there are
no successful inhibitors of this kind. Moreover,
there are only a few studies about the effects of
perylene derivatives on glutathione transferase
and various kinases. In this study, four novel per-
ylene compounds, N,N¢-disubstituted perylenedii-
mides and their 1,7-dibromo derivatives, were
synthesized and evaluated for their biological
activities. Here, among the compounds analyzed,
one of them was identified with strong glutathi-
one transferase inhibition and two with dual activ-
ity for both glutathione transferase and c-src
inhibition. These results revealed that perylene
derivatives may be employed as potential chemo-
sensitizers to prevent chemotherapy-dependent
drug resistance and identified as prospective anti-
cancer agents with dual activity on both glutathi-
one transferase and c-src enzymes.
Key words: anticancer agent, glutathione transferase, perylenedii-
mide, small-molecule inhibitor, tyrosine kinase
Received 22 January 2012, revised 12 June 2012 and accepted for pub-
lication 16 July 2012
Perylene derivatives are compounds used in a variety of industrial
applications for decades, especially in the dye-sensitized solar
1

Keskin et al.
cancer cell lines (18–22). However, such studies, searching the
biological effects, were focused mainly on cytotoxic effects of per-
ylene derivatives on cell viability, without mentioning the signaling
components that contribute to the therapeutic effectiveness of
compounds. In signal transduction from extracellular stimuli
through cell membrane to cytoplasm, and to the nucleus, Src
kinase (Src), among the PTKs, emerges as a fundamental constitu-
ent with essential roles in diverse signaling mechanisms (23).
Therefore, the deregulations in its activity have been associated
with various pathologic conditions, including cancer. In this con-
text, studies with hypericin in photodynamic therapy applications
revealed its association with GST and various members of PTK,
where the enhanced photodynamic response was reported with
GST inhibition at low nanomolar ranges of compound adminis-
tered, and related with the decreased GST-dependent resistance
(19–22).
with absorption peaks beyond 650 nm as an alternative photosen-
sitizer for photodynamic therapy (24). With these compounds, we
demonstrated that the cytotoxicity of derivatives on red-light exci-
tation was more significant compared to non-excited compounds.
These preliminary in vitro experiments, hence, verified their poten-
tial utility in photodynamic therapy. Consistent with these efforts,
in this current study, we report the design and synthesis of novel
perylenediimides (Scheme 1: panel A) with amino acid substitu-
tions (Scheme 1: panel B) and their inhibitory characteristics
against two critical enzyme targets. To reveal their potential to
minimize tissue resistance while improving anticancer effective-
ness, the structure-activity relationships were assessed by dose–
response studies.
Experimental Section
Perylenediimides are known for their photo- and chemical stability.
However, they do not have absorptions in the red end of the visi-
ble spectrum to be suitable for photodynamic applications. Our
previous efforts to design and develop novel perylenediimide
derivatives resulted in compounds with potential efficacy in photo-
dynamic therapy (24–26). Recently, green perylenediimides with
dialkylamino substituents on the perylene core was reported as
an alternative photosensitizer for photodynamic therapy (27). Previ-
ously, we synthesized water-soluble green perylenediimide dyes
Chemistry
L-glutamic acid di-tert-butylester hydrochloride, L-alanine tert-butyl-
ester hydrochloride, n-butanol, and the tyrosine kinase inhibitors
Genistein and SU6656 were purchased from Sigma-Aldrich, St.
Louis, MO, USA. 3,4:9,10-perylenetetracarboxylic acid 3,4:9,10-dian-
hydride, sulfuric acid, triethylamine, acetic acid, trifluoroacetic acid,
liquid Br2, silica gel 60 (particle size: 0.040–0.063 mm, 230–400
mesh ASTM), silica gel 60 Kiesel gel F254 TLC Aluminum Sheets
were purchased from Merck, Darmstadt, Germany. Takara Universal
Scheme 1: Panel A- the synthesis of N,N¢-di-
substitutedperylenediimides and their 1,7-dibromo
derivatives. Panel B- structures of hypericin and
the synthesized novel erylenediimide derivatives.
2
Chem Biol Drug Des 2012

Kinase and Transferase Activity of Perylenediimides
Tyrosine Assay ELISA plates and reagents were purchased from Ta-
kara-Bio Inc., Shiga, Japan; recombinant Src and Profluor Fluores-
cent Tyrosine Kinase reagents were purchased from Promega,
Madison, WI, USA. Bovine liver cytosolic fraction was used as GST
enzyme source and prepared in our laboratory. Other chemicals
were at analytical grade and purchased from Sigma-Aldrich. The
nuclear magnetic resonance spectra (1H- and 13C-NMR) were
recorded using Avance Series-DX-400 (d given in ppm, J in Hz), Bru-
ker Instruments, Madison, WI, USA. All chemical shift values were
expressed in parts per million (ppm) relative to tetramethylsilane
(Me4Si) as an internal standard, and signals were reported as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Electro-
spray ionization (ESI) mass spectra were recorded on Agilent 6500
Series LC-MS spectrometer, Agilent instruments, Paolo Alto, CA,
USA. The kinetic and end-point detection to measure the substrate
phosphorylation or glutathione (GSH) conjugation was performed
with absorbance- and fluorescence-based assays and recorded by
SpectraMax-M2e Multi-Mode Microplate Reader, Molecular Devices
Corporation, Sunnyvale, CA, USA.
Synthesis of N,N¢-(L-glutamic acid tert-
butylester)-3,4:9,10-perylenediimide (I-b)
0.5 g (1.274 · 10)3 mol) perylene-3,4:9,10-tetracarboxylic acid dian-
hydride and 0.753 g (2.548 · 10)3 mol) glutamic acid di-tert-buty-
lester hydrochloride were dissolved in 10 mL H2O, 10 mL n-butanol
and 1.5 mL triethylamine and stirred for 48 h at 85 ꢀC. After sol-
vent removed, the product was purified via column chromatography
(trichloromethane ⁄ methanol, 97:3) and dried in vacuo (yield, 35%).
C50H54N2O12, ESI-MS: m ⁄ z 874.36 (M+ + 1).
1H NMR (400 MHz, CDCl3), d[ppm]: 1.39 (s, 36H, -CH3), 2.36 (m, 4H,
-CH2), 2.65 (m, 4H, -CH2), 5.65 (t, 2H, N-CH), 8.45 (d, J = 8.12 Hz,
4H, CH-arom), 8.55 (d, J = 9.6 Hz, 4H, CH-arom).
13C NMR (100 MHz, CDCl3), d[ppm]: 24.13, 27.97, 32.41, 53.83,
76.80, 81.71 (aliphatic), 122.96, 126.22, 129.36, 131.45, 134.41 (aro-
matic); 162.59 (1-amide); 167.97; 171.49 (C=O).
Synthesis of 1,7-dibromo-N,N¢-(L-alanine tert-
butylester)-3,4:9,10-perylenediimide (II-a)
Synthesis of 1,7-dibromo-3,6:9,10-
0.5 g (9.089 · 10)4 mol) 1,7-dibromo-perylenetetracarboxylic acid
3,4:9,10 dianhydride (II) and 0.33 g (1.817 · 10)3 mol) alanine tert-
butylester hydrochloride were dissolved in 10 mL H2O, 10 mL n-but-
anoland 1.5 mL triethylamine and stirred for 48 h at 85 ꢀC. After sol-
vent removed, the product was purified via column chromatography
(trichloromethane ⁄ methanol, 98:2), and dried in vacuo (yield, 30%).
perylenetetracarboxylic acid dianhydride (II)
A mixture of 1.47 g (3.74 · 10)3 mol) perylene-3,4:9,10-tetracarb-
oxylic acid dianhydride (I), 12.01 mL H2SO4 and 0.032 g
(1.26 · 10)4 mol) I2 was stirred for 12 h at room temperature (RT)
and heated to 85 ꢀC for 30 min. After cooling to RT, 2.1 mL Br2
was added to the mixture, in the pressure tube, within 8 h. The
mixture was heated to 85 ꢀC, and then, 3 mL Br2 was added for a
period of 12 h. The concentration of H2SO4 was decreased to 86%
by adding 1.67 mL of water in 1 h. Finally, the mixture was cooled
to RT, filtered under suction through a G-4 glass-frit filtering cruci-
ble, washed with 15 mL H2SO4 (86% w ⁄ w), and then with H2O.
The bright-red precipitate was dried in a vacuum oven at 120 ꢀC
for 24 h (yield, 82%).
C36H28Br2N2O8, ESI-MS: m ⁄ z 804.049 (M+ + 1).
1H NMR (400 MHz, CDCl3), d[ppm]: 1.49 (s, 18H, -CH3), 1.67 (d,
J = 7.09 Hz, -CH3), 5.22 (m, 2H, N-CH), 8.71 (d, J = 8.161 Hz, 4H,
CH-arom), 8.92 (s, 2H, CH-arom), 9.5 (d, J = 8.16 Hz, 4H, CH-arom).
13C NMR (100 MHz, CDCl3), d[ppm]: 24.06, 29.74, 60.74, 81.11 (ali-
phatic), 117.82, 123.18, 128.43, 132.84 (aromatic), 168.30 (1-amide),
184.64 (C=O).
C24H6Br2O6.
Synthesis of 1,7-dibromo-N,N¢-(L-glutamicacid
tert -butylester)-3,4:9,10-perylenediimide (II-b)
0.5 g (9.089 · 10)4 mol) 1,7-dibromo-perylenetetracarboxylic acid
3,4:9,10 dianhydride (II) and 0.537 g (1.817 · 10)3 mol) alanine tert-
butylester hydrochloride were dissolved in 10 mL H2O, 10 mL n-buta-
nol and 1.5 mL triethylamine and stirred for 48 h at 85 ꢀC. After sol-
vent removed, the product was purified via column chromatography
(trichloromethane ⁄ methanol, 95:5) and dried in vacuo (yield, 32%).
Synthesis of N,N¢-(L-alanine tert-butylester)-
3,4:9,10-perylenediimide (I-a)
0.5 g (1.274 · 10)3 mol) perylene-3,4:9,10-tetracarboxylic acid dian-
hydride and 0.462 g (2.548 · 10)3 mol) alanine tert-butylester
hydrochloride were dissolved in 10 mL H2O, 10 mL n-butanol and
1.5 mL triethylamine and stirred for 48 h at 85 ꢀC. After solvent
removed, the product was purified via column chromatography (tri-
chloromethane ⁄ methanol, 97:3), and dried in vacuo (yield, 32%).
C36H30N2O8, ESI-MS: m ⁄ z 646.22 (M+ + 1).
C50H52Br2N2O12, ESI-MS: m ⁄ z 1032.18.
1H NMR (400 MHz, CDCl3), d[ppm]: 1.36 (s, 36H, -CH3), 2.3 (m, 2H,
-CH2), 2.7 (m, 2H, -CH2), 5.6 (t, 2H, N-CH), 8.7 (d, J = 8.16 Hz, 2H,
CH-arom), 8.9 (s, 2H, CH-arom), 9.5 (d, J = 8.16 Hz, 2H, CH-arom).
1H NMR (400 MHz, CDCl3), d[ppm]: d 1.52 (s, 18H, -CH3), 1.71 (d,
J = 7 Hz, -CH3), 5.68 (m, 2H, N-CH), 8.35 (d, J = 8.08 Hz, 4H, CH-
arom), 8.49 (d, J = 7.96 Hz, 4H, CH-arom).
13C NMR (100 MHz, CDCl3), d[ppm]: 29.26, 30.18, 32.31, 45.30,
80.51 (aliphatic), 127.45, 130.18, 137.04, 138.12, 144.26 (aromatic),
167.59 (1-amide), 168.73, 171.38 (C=O).
13C NMR (100 MHz, CDCl3), d[ppm]: 14.79 (aliphatic), 27.96, 29.60,
50.15, 81.47, 122.72, 123.09, 125.99, 129.10, 131.20, 134.17
(aromatic), 162.15 (1-amide), 168.57 (C=O).
Chem Biol Drug Des 2012
3

Keskin et al.
Evaluation of perylene derivatives through
glutathione transferase assays
each microplate well is inversely related to the kinase activity of
the enzyme within the wells and comparison performed with
respect to control wells. This assay also monitors the AMC fluores-
cent signal, which is the coumarin derivative without phosphoryla-
tion site available, to evaluate whether the observed kinase
inhibition results are false positive or not. The IC50 values of com-
pounds were determined by comparing the Src activity of the wells
containing compounds, with the activities of vehicle and positive
control (genistein and SU6656) wells.
The slightly modified method of Habig (28) was used to measure
the kinetic change in substrate (GSH) utilization by GST. Then, the
method was adopted for microscale applications. Briefly, the total
GST activity was measured using reaction buffer (100 mM potassium
phosphate buffer at pH 6.5 containing 2.4 mM CDNB and 3.2 mM
GSH) (29,30). In the assay, the compounds were incubated with
reaction buffer at RT (22–25 ꢀC) for 5 min. Upon addition of enzyme
(bovine liver cytosol), the plate was mixed for 30 seconds, and
GSH-CDNB conjugate formation, as an increase in the absorbance,
was monitored at 340 nm for 240 seconds. Initial rates of enzy-
matic reactions were determined for the formation of GSH-conjuga-
tion product, and the activity of enzyme samples was determined
as nmol ⁄ minute ⁄ mg protein, where the total protein content was
determined by the Lowry method. For the analysis of compounds,
the kinetic measurements were performed with compounds dis-
solved in dimethyl sulfoxide (DMSO, 0.5% per assay volume), and
the calculations were made with respect to vehicle control (the
assay buffer with 0.5% DMSO) without compounds. The maximum
inhibition was determined using 100 mM of ethacrynic acid as posi-
tive control. The dose–response curves were constructed using the
absorbance change (enzyme activity in the assay) from the kinetic
data and reported as OD340 ⁄ min. All measurements were performed
in 96-well microplate, using Spectramax M2e. IC50 values were
determined as the concentration of a compound required to achieve
50% inhibition of enzyme activity with respect to the vehicle control
and determined by nonlinear regression analysis, the four parameter
logistic equation (Sigmoidal dose–response, GRAPHPAD PRISM version
4.0 for Windows, GRAPHPAD Software, San Diego, CA, USA).
Results and Discussion
The drawbacks in cancer research mainly because of the cellular
resistance to chemotherapeutics and the toxic side-effects of drugs.
Therefore, to develop therapeutics with full biological response, it
appears critical to evaluate their interactions with drug metabolizing
enzymes, such as GSTs, and their possible contribution to PTK-medi-
ated signaling mechanisms, such as Src. Depending on duration,
dosage, and chemical composition of chemotherapeutics adminis-
tered, the acquired resistance is commonly experienced problem
and associated with the overactive GST isozymes. In an attempt to
overcome this difficulty, in particular linked to systemic toxicity, vari-
ous compounds that possibly modify the activities of detoxification
system enzymes have been explored. For this purpose, we described
the synthesis and chemical characterization of 3,6:9,10-perylenedii-
mide derivatives as new compounds to be developed for selective
transferase and kinase inhibitors. The required dibromo-substituted
perylenediimide II was synthesized similar to the practical and
straightforward synthesis with perylene-3,4:9,10-tetracarboxylic acid
dianhydride I as a starting material (26). Amino acid modifications
on the imide position of perylenedianhydride were considered to
improve the compound solubility in physiological medium and hence
the improved absorption by cells. In this context, the most circulat-
ing amino acids of human metabolism, namely alanine and glutamic
acid, were chosen for substitution. The reaction of tetracarboxylic
acid anhydride with either aliphatic or aromatic primary amines
was shown that the corresponding diimides were in good yield.
Similarly, tert-butylester derivative of amino acid with I or II
afforded perylenediimides with more than 30% yield. We have synthe-
sized four novel perylenediimides (Scheme 1: panel A): N,N¢-(L-alanine
tert-butylester)-3,4:9,10-perylenediimide (I-a), N,N¢-(L-glutamic acid tert-
butylester)-3,4:9,10-perylenediimide (I-b), 1,7-dibromo-N,N¢-(L-alanine
tert-butylester)-3,4:9,10-perylenediimide (Ia), and 1,7-dibromo-N,N¢-
(L-glutamic acid tert-butylester)-3,4:9,10-perylenediimide (Ib), where
tert-butyl ester of L-alanine and di-tert-butyl ester of L-glutamic acid
were used as amine substituents. The chemical characterization of
Evaluation of perylene derivatives through in
vitro tyrosine kinase assays
The inhibition of the synthesized compounds was determined by vir-
tue of the ELISA based in vitro Tyrosine Kinase Assay (Takara Uni-
versal Protein Tyrosine Kinase Assay, Takara Corp., Tokyo, Japan)
and fluorescent dye Rhodamine 110 (R110) based indirect phosp-
hotyrosine phosphatase based kinase assay (Profluor; Promega Inc.,
Madison, WI, USA). The kinase assay was performed at 37 ꢀC, with
40 nM ATP (final assay volume of 50 lL). The enzyme activity was
probed with HRP-conjugated anti-phosphotyrosine (PY20) antibody
at 450 nm (31). The ELISA assay was used to determine the 80%
active Src concentration for fluorescent end-point detection of
kinase activity. The activities of the Src to construct the calibration
curve were as follows: 1520, 760, 380, 190, 94.8, and 47.4 · 10)7
,
units ⁄ lL. Then, the inhibitory activities of compounds were moni-
tored by fluorescent end-point detection, that is, ProFluor Assay Pro-
tocol, with some modifications (31). In this assay, the pure enzyme
Src (Promega) was used to achieve 20% of the maximum fluores-
cent signal (80% active enzyme) and the compounds were prepared
at final concentrations of 4.50 mM to 0.10 lM in DMSO. Briefly, the
molecules were mixed with Src-family kinase R110 substrate, and
the reaction was initiated with the addition of ATP, followed by
incubation at 22 ꢀC for 60 min. The protease solution was added,
and mixture was incubated another 60 min at RT. The reaction was
terminated with protease inhibitor, and the fluorescence of R110
was read at 525 nm (Ex 460 nm). The decrease in fluorescence of
1
compounds was verified by H- and 13C-NMR and MS (ESI) analysis,
and the spectral properties were investigated in chloroform. The
tert-butylester groups of amino acids were removed before biologi-
cal assays to improve the solubility of compounds in aqueous buf-
fers because of the nature of amino acid substituents and in
common organic solvents because of the perylene core. The tert-
butyl protection on propionic (alaninyl) and pentanedioic acid (glut-
amyl) functions on N atom were removed by trifluoroacetic-
acid ⁄ chloroform treatment (Scheme 1, panel A,B). Then, the
compounds (Ia–IIb) were evaluated for their inhibitory activities
toward tyrosine phosphorylation of Src and S-glutathionylation of
4
Chem Biol Drug Des 2012

Kinase and Transferase Activity of Perylenediimides
GST. Similar to our previous approach, we also investigated the
effects of substituents at bay position that is the region located
perylene core scaffold and the amino acid substitution to form
piperidine from pyran ring. The core compounds I and II did not
show any inhibition profile, where the IC50 values were not assess-
able. Compounds IIa and IIb turned out to be strong inhibitors of
GST with IC50 values of 106.3 € 27.3 (Figure 1) and 709.4 €
23.6 nM (Figure 2), respectively, and are more effective than com-
pound Ia with IC50 of 2.044 € 0.032 lM (Figure 3). These important
differences in the inhibitory potencies and profiles were possibly
due to the presence of bromide in the bay region of perylene core
and considered to increase the GST affinity toward these molecules.
Notably, Ia (Figure 4) and IIb (Figure 5) showed inhibition of Src
with IC50 values of 433.1 € 11.9 mM and 632.3 € 66.8 lM, respec-
tively. The compounds Ib and IIa showed very low inhibition
against Src (IC50 > 1 mM), where the dose–response curves did not
fit 4-parameter logistic equation (data not shown). Neither changes
in the final concentrations in assay, nor the alterations in reaction
conditions resulted in reasonable effect of Ib or IIa on target
enzyme activities. This is the first demonstration of a possible anti-
cancer activity of the novel perylenediimide derivatives with amino
acid functional groups, without irradiation, and the first report in
investigating their effects on Src, and cytosolic GSTs. As the GSTs
are highly overexpressed in cancer cells, their inhibition by peryl-
enediimides might offer new clues on their mechanism of action
and open interesting perspectives for future tumor therapies.
Figure 3: Dose–response curve for glutathione transferase
enzyme activity (OD340 ⁄ min) against varying doses of compound Ia,
where the IC50 value is 2.044 € 0.032 lM.
Figure 1: Dose–response curve for glutathione transferase
enzyme activity (OD340 ⁄ min) against varying doses of compound IIa,
where the IC50 value is 106.3 € 27.3 nM.
Figure 2: Dose–response curve for glutathione transferase
enzyme activity (OD340 ⁄ min) against varying doses of compound IIb,
where the IC50 value is 709.4 € 23.6 nM.
Figure 4: Dose–response curve for Src enzyme activity pre-
sented as change in fluorescence against varying doses of com-
pound Ia, where the IC50 is 433.1 € 11.9 lM.
Chem Biol Drug Des 2012
5

Keskin et al.
6. Kubin A., Wierrani F., Burner U., Alth G., Grunberger W. (2005)
Hypericin – the facts about a controversial agent. Curr Pharm
Des;11:233–253.
7. Agostinis P., Berg K., Cengel K.A., Foster T.H., Girotti A.W., Goll-
nick S.O., Hahn S.M. et al. (2011) Photodynamic therapy of can-
cer: an update. CA Cancer J Clin;61:250–281.
8. Boiy A., Roelandts R., de Witte P.A. (2010) Photodynamic therapy
using topically applied hypericin: comparative effect with
methyl-aminolevulinic acid on UV induced skin tumours. J Photo-
chem Photobiol, B;102:123–131.
9. Gulick A.M., Fahl W.E. (1995) Forced evolution of glutathione S-
transferase to create a more efficient drug detoxication enzyme.
Proc Natl Acad Sci U S A;92:8140–8144.
10. Isgor Y.G., Isgor B.S. (2011) Kinases and glutathione transfer-
ases: selective and sensitive targeting. Front Biol;6:156–169.
11. Di Pietro G., Magno L., Rios-Santos F. (2010) Glutathione S-
transferases: an overview in cancer research. Expert Opin Drug
Metab Toxicol;6:153–170.
Figure 5: Dose–response curve for Src enzyme activity pre-
sented as change in fluorescence against varying doses of com-
pound IIb, where the IC50 is 632.3 € 66.8 lM.
12. Hayes J.D., Flanagan J.U., Jowsey I.R. (2005) Glutathione trans-
ferases. Annu Rev Pharmacol Toxicol;45:51–88.
13. Okamura T., Singh S., Buolamwini J., Haystead T., Friedman H.,
Bigner D., Ali-Osman F. (2009) Tyrosine phosphorylation of the
human glutathione S-transferase P1 by epidermal growth factor
receptor. J Biol Chem;284:16979–16989.
Acknowledgments
14. Hosono N., Kishi S., Iho S., Urasaki Y., Yoshida A., Kurooka H.,
Yokota Y., Ueda T. (2010) Glutathione S-transferase M1 inhibits
dexamethasone-induced apoptosis in association with the sup-
pression of Bim through dual mechanisms in a lymphoblastic
leukemia cell line. Cancer Sci;101:767–773.
This work was partially supported by grants from the Atilim Univer-
sity (BAP 2011), Balikesir University (BAP2009 ⁄ 40; BAP 2010 ⁄ 18),
The Scientific and Technological Research Council of Turkey (TUBI-
TAK-110T026), and Undersecretariat of State Planning Organization
(DPT-2005-K-120-170).
15. Singh S., Okamura T., Ali-Osman F. (2010) Serine phosphorylation
of glutathione S-transferase P1 (GSTP1) by PKCalpha enhances
GSTP1-dependent cisplatin metabolism and resistance in human
glioma cells. Biochem Pharmacol;80:1343–1355.
Conflict of Interest
The authors have declared no conflict of interest.
16. Desmots F., Rissel M., Gilot D., Lagadic-Gossmann D., Morel
F., Guguen-Guillouzo C., Guillouzo A., Loyer P. (2002) Pro-
inflammatory cytokines tumor necrosis factor alpha and inter-
leukin-6 and survival factor epidermal growth factor positively
regulate the murine GSTA4 enzyme in hepatocytes. J Biol
Chem;277:
References
1. Jin Y., Hua J., Wu W., Ma X., Meng F. (2008) Synthesis, charac-
terization and photovoltaic properties of two novel near-infrared
absorbing perylene dyes containing benzo[e]indole for dye-sensi-
tized solar cells. Synth Met;158:64–71.
2. Zafer C., Kus M., Turkmen G., Dincalp H., Demic S., Kuban B.
(2007) New perylene derivative dyes for dye-sensitized solar
cells. Sol Energy Mater Sol Cells;91:427–431.
3. Chiu T.L., Chuang K.H., Lin C.F., Ho Y.H., Lee J.H., Chao C.C.
(2009) Low reflection and photo-sensitive organic light-emitting
device with perylenediimide and double-metal structure. Thin
Solid Films;517:3712–3716.
4. Wang X., Guo Y., Yang S., Wang C., Fu X., Wang J., Mao Y.,
Zhang J., Li Y. (2010) Cellular and molecular mechanisms of
photodynamic hypericin therapy for nasopharyngeal carcinoma
cells. J Pharmacol Exp Ther;334:847–853.
17892–17900.
17. Kim S.G., Lee S.J. (2007) PI3K, RSK, and mTOR signal
networks for the GST gene regulation. Toxicol Sci;96:206–213.
18. Chan P.S., Koon H.K., Wu Z.G., Wong R.N., Lung M.L., Chang
C.K., Mak N.K. (2009) Role of p38 MAPKs in hypericin photody-
namic therapy-induced apoptosis of nasopharyngeal carcinoma
cells. Photochem Photobiol;85:1207–1217.
19. Du H.Y., Olivo M., Tan B.K., Bay B.H. (2004) Photoactivation of
hypericin down-regulates glutathione S-transferase activity in
nasopharyngeal cancer cells. Cancer Lett;207:175–181.
20. Dabrowski M.J., Maeda D., Zebala J., Lu W.D., Mahajan S.,
Kavanagh T.J., Atkins W.M. (2006) Glutathione S-transferase P1-
1 expression modulates sensitivity of human kidney 293 cells to
photodynamic therapy with hypericin. Arch Biochem Bio-
phys;449:94–103.
5. Krishnamoorthy G., Webb S.P., Nguyen T., Chowdhury P.K.,
Halder M., Wills N.J., Carpenter S., Kraus G.A., Gordon M.S.,
Petrich J.W. (2005) Synthesis of hydroxy and methoxy perylene
quinones, their spectroscopic and computational characterization,
and their antiviral activity. Photochem Photobiol;81:924–933.
21. Chang Y., Wang S.J. (2010) Hypericin, the active component of
St. John's wort, inhibits glutamate release in the rat cerebrocor-
tical synaptosomes via a mitogen-activated protein kinase-
dependent pathway. Eur J Pharmacol;634:53–61.
6
Chem Biol Drug Des 2012

Kinase and Transferase Activity of Perylenediimides
22. Tuna G., Kulaksiz Erkmen G., Dalmizrak O., Dogan A., Ogus I.H., Ozer
N. (2011) Inhibition characteristics of hypericin on rat small intes-
tine glutathione-S-transferases. Chem Biol Interact;188:59–65.
23. Belsches-Jablonski A.P., Demory M.L., Parsons J.T., Parsons S.J.
(2005) The Src pathway as a therapeutic strategy. Drug Discov
Today;2:313–321.
24. Yukruk F., Dogan A.L., Canpinar H., Guc D., Akkaya E.U. (2005)
Water-soluble green perylenediimide (PDI) dyes as potential sen-
sitizers for photodynamic therapy. Org Lett;7:2885–2887.
25. Yukruk F., Akkaya E.U. (2005) Modulation of internal charge
transfer (ICT) in a bay region hydroxylated perylenediimide (PDI)
chromophore: a chromogenic chemosensor for pH. Tetrahedron
Lett;46:5931–5933.
27. Qu J., Kohl C., Pottek M., Mꢀllen K. (2004) Ionic perylenetetra-
carboxdiimides: highly fluorescent and water-soluble dyes for bi-
olabeling. Angew Chem Int Ed;43:1528–1531.
28. Habig W.H., Jakoby W.B. (1981) Glutathione S-transferases (rat
and human). Methods Enzymol;77:218–231.
29. Geylan-Su Y.S., Isgor B., Coban T., Kapucuoglu N., Aydintug S.,
Iscan M., Guray T. (2006) Comparison of NAT1, NAT2 and
GSTT2-2 activities in normal and neoplastic human breast tis-
sues. Neoplasma;53:73–78.
30. Isgor B.S., Coruh N., Iscan M. (2010) Soluble glutathione S-
transferases in bovine liver: existence of GST T2. J Biol Sci;10:
667–675.
31. Isgor B.S., Isgor Y.G. (2012) Effect of alpha-1-adrenoceptor
blocker on cytosolic enzyme targets for potential use in cancer
chemotherapy. Int J Pharm;8:333–343.
26. Yukruk F. (2006) Optical fluoride sensing with a Bay region func-
tionalized perylenediimide dye. Turk J Chem;30:455–460.
Chem Biol Drug Des 2012
7