(E)-2-Benzylidenebenzocyclanones: Part VII. Investigation of the conjugation reaction of two cytotoxic cyclic chalcone analogues with glutathione: An HPLC-MS study

Article abstract of DOI:10.1007/s00706-012-0768-7

The non-enzyme-catalyzed reaction of reduced glutathione (GSH) with two tumor cell cytotoxic cyclic chalcone analogues was investigated by reversed-phase high-performance liquid chromatography (RP-HPLC). HPLC analysis of the reaction mixtures indicated th

Full text of DOI:10.1007/s00706-012-0768-7

Monatsh Chem (2012) 143:1107–1114
DOI 10.1007/s00706-012-0768-7
ORIGINAL PAPER
(E)-2-Benzylidenebenzocyclanones: Part VII. Investigation
of the conjugation reaction of two cytotoxic cyclic chalcone
analogues with glutathione: an HPLC–MS study
•
Pal Perjesi Gabor Maasz Renata Reisch
•
•
´
Andras Benko
´
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´
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}
Received: 28 September 2011 / Accepted: 11 April 2012 / Published online: 7 June 2012
Ó Springer-Verlag 2012
Abstract The non-enzyme-catalyzed reaction of reduced
glutathione (GSH) with two tumor cell cytotoxic cyclic
chalcone analogues was investigated by reversed-phase
high-performance liquid chromatography (RP-HPLC).
HPLC analysis of the reaction mixtures indicated the for-
mation of two diastereomeric chalcone–GSH adducts in
each case, whose structural assignments were supported by
MALDI-TOF-MS and HPLC–MS with electrospray ioni-
zation (ESI) measurements. Such reactivity accounts for the
previously observed effect of the two cyclic chalcone ana-
logues on the in vivo cellular thiol level of Jurkat T cells.
and antitumor promoting, anti-inflammatory, and chemo-
preventive agents [3–5].
Nucleophilic addition of cellular thiols onto the polar-
ized carbon–carbon double bond (Michael reaction) is
frequently associated with the biologic effects of chalcones
and other a,b-unsaturated carbonyl compounds [3, 4]. Such
a reaction can alter the intracellular redox status (redox
signaling), which can modulate events such as DNA syn-
thesis, enzyme activation, selective gene expression, and
regulation of the cell cycle [6]. Several biological effects
(e.g., NQO1 inducer [7], anti-inflammatory [8], and GST
P1-1 inhibitory [9] effects) of chalcones have been asso-
ciated with their Michael-type reactivity towards reduced
glutathione (GSH). It was suggested that the lower GSH-
depletion potential of chalcones with strong electron donor
substituents (e.g., dimethylamino) on the B ring could be
the consequence of the lower Michael-type reactivity of the
derivatives towards GSH [8]. On the other hand, higher
reactivity towards GSH and other thiols was found to
parallel the higher NQO1 inducing potential of the inves-
tigated chalcones [7].
Keywords Cyclic chalcone analogue Á Enones Á
Glutathione Á Michael addition Á Bioorganic chemistry
Introduction
Chalcones (1, Fig. 1) are intermediates of the biosynthetic
pathway of a very large and widespread group of plant
constituents known collectively as flavonoids [2]. Among
the naturally occurring chalcones and their synthetic ana-
logues, several compounds displayed cytotoxic (cell growth
inhibitor) activity towards cultured tumor cells. Chalcones
are also effective in vivo as cell proliferating inhibitors
Recently we investigated the in vitro antineoplastic
activity of several synthetic chalcones and cyclic chalcone
analogues of different ring size. The compounds were
evaluated against P388, L1210, Molt 4/C8, and CEM cells
as well as against approximately 60 further human tumor
cell lines from nine different neoplastic diseases [10–12].
Among the compounds investigated the seven-membered
cyclic chalcone analogue (E)-2-(4-methoxybenzylidene)-1-
benzosuberone (2, Fig. 1) had the greatest tumor toxicity. It
was demonstrated that compound 2 and its methyl-substi-
tuted analogue 3 can modify the free thiol content of the
investigated Jurkat T cells [13]. The compounds and their
structural analogues have been found to modify the GSH
content of rat liver mitochondria as well [14]. These results
For part VIII in this series, see ref. [1].
´
}
P. Perjesi (&) Á R. Reisch Á A. Benko
´
Department of Pharmaceutical Chemistry, University of Pecs,
´ ´
Rokus u 2, 7634 Pecs, Hungary
e-mail: pal.perjesi@aok.pte.hu
´
G. Maasz
Department of Biochemistry and Medical Chemistry,
´ ´
University of Pecs, Szigeti u 12, 7634 Pecs, Hungary
123

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P. Perjesi et al.
1108
carbon double bonds mainly by nucleophilic attack of their
thiolate anion [17]. The concentration of the reactive
nucleophile is determined by the pKa, which is about 9.2
for GSH [18]. Earlier results demonstrated that under
slightly acidic conditions—characteristic for tumor cells—
compounds 2 and 3 did not show intrinsic reactivity with
GSH at 37 °C [13]. In the present study, the pH of the
reaction mixtures was set to 8.0 at which the much stronger
nucleophilic thiolate anions represent 6 % of total GSH.
Non-enzyme-catalyzed reaction of 2 and 3 with GSH
was expected to result in the formation of two diastereo-
meric adducts (conjugates 1: 2a, 3a and conjugates 2: 2b,
3b) as a result of cis and trans addition of GSH onto the
carbon–carbon double bond (Fig. 2). Earlier formation of
such a diastereomeric pair of adducts was observed in the
glutathione-S-transferase (GST)-catalyzed reaction of GSH
and the open-chain chalcone analogue (E)-4-(4-X-phenyl)-
3-butene-2-ones [19].
O
O
X
1
X = OCH3
X = CH3
2
3
Fig. 1 Chemical structures of chalcone (1), (E)-2-(4-methoxybenz-
ylidene)-1-benzosuberone (2), and (E)-2-(4-methylbenzylidene)-1-
benzosuberone (3)
were in accordance with the reported reactivity of GSH
with the investigated chalcones [13, 14].
As a continuation of our work towards a better under-
standing of the molecular mechanisms taking part in the
development of biological effects of chalcones, we studied
the non-enzyme-catalyzed reaction of (E)-2-(4-methoxy-
benzylidene)-1-benzosuberone (2) and (E)-2-(4-methyl-
benzylidene)-1-benzosuberone (3) with GSH. Our primary
goal was to investigate the intrinsic (non-enzyme-catalyzed)
reactivity of the compounds towards GSH, characterize the
structure of the products, and develop an HPLC method to
detectboththeparentcompounds and theirGSH conjugatesin
vitro and in vivo.
Initially, the progress of the reactions was monitored by
UV–Vis measurements. Electronic transitions in the (E)-2-
benzylidene-1-benzocyclanones afforded two absorption
bands in the UV region in the spectra generally denoted as
band I (300–400 cm-1) and band II (200–300 cm-1),
respectively [20]. In the case of flavones band I is associ-
ated with the cinnamoyl moiety and band II with the
benzoyl moiety of the compounds, both involving p–p*
electron transitions [21]. The progress of the addition
reactions was monitored by measurement of the band I
absorption (332 and 315 nm for 2 and 3, respectively) of
the starting chalcones 2 and 3. At 300 min the absorption
of 2 was reduced to 83 %, whereas that of 3 was 70 % of
the respective initial values. This observation is in agree-
ment with the expected stability of the adducts which is
affected by the different degree of electron donation of the
two substituents [8].
Results and discussion
The effect of the cyclic chalcone analogues 2 and 3 on the
cell cycle of Jurkat T cells was investigated previously
[13]. It was found that equitoxic doses of the two com-
pounds (5.6 lM (2) and 23.3 lM (3)) displayed different
effects on the cell cycle and modified the free cellular thiol
status of the cells. Since GSH represents almost the whole
free cellular thiols, the reaction of GSH with the cyclic
chalcone analogues 2 and 3 was investigated in cell-free
conditions using a tenfold molar excess of GSH.
HPLC analysis of the composition of the reaction mix-
tures was performed by gradient RP-HPLC using UV
detection at 210 and 260 nm. Thus, the selected wave-
lengths were characteristic for not only the starting 2 and 3
but also for their GSH adducts (2a, 2b and 3a, 3b) as well.
The method developed provided baseline separation of the
starting chalcones and the formed GSH conjugates. The
HPLC–UV chromatograms (k = 260 nm) of the reaction
GSH is known to form S-linked conjugates with a,b-
unsaturated carbonyl compounds via 1,4-Michael reaction.
Although such thiol additions are, in principle, chemically
reversible [15], GSH conjugates of a,b-unsaturated car-
bonyl compounds appear, in general, to be relatively stable
compounds [16]. Thiols react with polarized carbon–
Fig. 2 Reaction between
SG
O
O
reduced glutathione (GSH) and
the cyclic chalcone analogues
2 and 3
+ GSH
X
X
conjugate 1 conjugate 2
2
3
2a
3a
2b
3b
X = OCH3
X = CH3
123

Conjugation reaction of two cytotoxic cyclic chalcone analogues with glutathione
1109
A
DAD1 A, Sig=260,4 Ref=600,100 (OE\OE1 2012-01-09 14-10-07\Q510.D)
mAU
175
2
150
125
100
2a
2b
75
50
25
0
0
5
10
15
20
25
min
B
DAD1 A, Sig=260,4 Ref=600,100 (OE\OE1 2012-01-09 15-06-50\Q511.D)
mAU
140
3
120
100
80
60
40
20
0
3b
3a
0
5
10
15
20
25
min
Fig. 3 HPLC–UV chromatograms (k = 260 nm) of the reaction mixtures of compounds 2 (a) and 3 (b) with GSH after 300 min incubation time
mixtures of 2 and 3 after 300 min incubation time are
depicted in Fig. 3a, b, respectively.
spectra of compounds 2 and 3 recorded in positive ion
mode and those of their respective incubation mixtures
(300 min incubation time) are depicted in Figs. 4 and 5,
respectively.
As shown in Fig. 3a, b, besides the respective starting
compounds 2 and 3—which were identified by standard
injection and ESI-MS—two new chromatographic peaks
corresponding to 2a, 2b (Fig. 3a) and 3a, 3b (Fig. 3b)
appear, indicating the formation of the expected two dia-
stereomeric GSH adducts of compounds 2 and 3. Similar
results were obtained by thin-layer chromatographic (TLC)
investigations of the reaction mixtures in which a com-
bined detection method (UV illumination plus ninhydrin
staining) also supported the formation of the expected
conjugates [13].
As shown, the protonated molecular peak of 2
(m/z = 279.397) and 3 (m/z = 263.443) as well as that of
their protonated ([M ? H]?) GSH adducts—m/z = 587.182
(average isotopic peak) and m/z = 570.227 (monoisotopic
peak), respectively—could be clearly identified in the spectra.
Besides the molecular peaks, characteristic fragments formed
by losses of pyroglutamic acid (129 Da) (2: m/z = 457.251;
3: m/z = 441.334), and pyroglutamic acid plus glycine
(204 Da) (2: m/z = 382.320; 3: m/z = 366.325) of the GSH
moiety of the conjugates can be identified in the spectra.
Laser-induced formation of these fragments is supported by
the fragmentation pathways reportedfor GSH conjugateswith
related structures [24–26].
Reaction of the open-chain chalcone analogues (E)-4-(4-
X-phenyl)-3-butene-2-ones with GSH also resulted in the
formation of two diastereomeric adducts [19]. Similarly,
investigation of the stereochemistry of the acid-catalyzed
addition of dithiocarbamic acid (another S-nucleophilic
reagent) onto the cyclic chalcone analogues (E)-2-benzy-
lidene-1-tetralone and -1-benzosuberone also showed the
formation of two diastereomeric adducts, as indicated by
1H NMR analysis of the crude products [22]. The stereo-
chemistry of the dithiocarbamic acid adduct of the five-
membered ring analogue (E)-2-benzylidene-1-indanone
was determined by X-ray diffraction [23].
Structural verification of the formed adducts was also
performed by a combination of the HPLC method with
electrospray ionization mass spectrometry (ESI-MS). The
HPLC–UV (k = 212 nm) and the HPLC–ESI-MS chro-
matograms of compounds 2 and 3 as well as those of their
GSH conjugates are shown in Figs. 6a–d and 7a–d,
respectively. The selected ion chromatograms (Figs. 6b,
7b) gave evidence of the formation of two diastereomeric
GSH adducts of 2 and 3 with the protonated molecular
masses of 586.182 and 570.227, respectively. Mass spectra
of the reaction mixtures recorded in the 50–2,000 Da range
The structures of the expected GSH adducts were
confirmed by MALDI-TOF-MS and HPLC–ESI-MS mea-
surements of the reaction mixtures. MALDI-TOF mass
123

´
P. Perjesi et al.
1110
x10
279.397
Q510\0_P18\1\1Ref
1.0
0.8
0.6
0.4
0.2
0.0
5
x10
Q510+GSH\0_P19\1\1SRef
279.387
1.25
1.00
0.75
0.50
0.25
0.00
382.320
587.182
457.251
450
587.182
600
250
300
350
400
500
550
m/z
Fig. 4 Positive ion MALDI-TOF spectra of compound 2 (upper panel) and 300 min incubation of 2 with GSH (lower panel)
(Figs. 6c, 7c) show the respective protonated molecular
ions of 2 and 3 and those of their GSH conjugates. The
mass spectra recorded in the 230–570/560 Da range
(Figs. 6d, 7d) indicate the presence of fragments of the
respective GSH conjugates that can be also identified in the
MALDI-TOF-MS spectra.
method coupled with ESI-MS detection provides the
appropriately selective and sensitive analytical tool to
investigate the chalcone–GSH reactions under cellular
conditions.
In conclusion, it was demonstrated that the cancer cell
cytotoxic cyclic chalcone analogs 2 and 3 possess inherent
reactivity with GSH to yield the expected diastereomeric
Michael adducts 2a, 2b and 3a, 3b. Such a reactivity,
fortified by the expected in vivo GST-catalyzed reactivity
[19], allows us to rationalize the previously observed effect
of the compounds on the cellular thiol level [13]. Depletion
of the cellular thiol level by xenobiotics or reactive elec-
trophilic metabolites can initiate a variety of effects in the
cells resulting in apoptosis and necrosis [27]. Accord-
ingly, the previously demonstrated apoptotic effect of
the two compounds [13] could be associated with their
(GST-catalyzed) reactivity towards GSH. Because of the
complex biochemical consequences of the reaction of
xenobiotics with cellular thiols [13], further studies are
needed to explore the role of GST-catalyzed reactivity of
the compounds in molecular mechanism of their recorded
tumor cell cytotoxicities. For such studies, the HPLC
Experimental
The cyclic chalcone analogues 2 and 3 were synthesized
and purified as described before [10]. Their structures were
characterized by IR and NMR spectroscopy [28]. Methanol
(HPLC grade) was produced by Carlo Erba (Milan, Italy).
Distilled water was purified by means of a Millipore
Direct-QTM system. Reduced glutathione (min. 99 %) and
trifluoroacetic acid (analytical grade) were purchased from
Sigma-Aldrich Hungary (Budapest, Hungary). Other chemi-
cals used were of the analytical grade available and, if
otherwise not specified, purchased from VWR International
(Debrecen, Hungary).
Reaction of compounds 2 and 3 with GSH
A solution of reduced glutathione (GSH, 1 cm3 of
2.0 9 10-1 M, 0.2 mmol) in distilled water (pH 8.0; 0.1 M
123

Conjugation reaction of two cytotoxic cyclic chalcone analogues with glutathione
1111
5
x10
Q511\0_J18\1\1SRef
263.443
1.0
0.8
0.6
0.4
0.2
0.0
4
x10
Q511+GSH\0_J19\1\1SRef
600.211
2.0
1.5
622.254
1.0
592.256
582.216
608.248
616.214
600.208
622.253
263.445
0.5
570.208
366.325
441.334
0.0
250
300
350
400
450
500
550
600
m/z
Fig. 5 Positive ion MALDI-TOF spectra of compound 3 (upper panel) and 300 min incubation of 3 with GSH (lower panel)
NaOH) was mixed with 3 cm3 of freshly prepared 6.67 9
10-3 M (0.02 mmol) methanol solution of 2 or 3. The final
concentration of GSH was 5 9 10-2 M and that of the
chalcones 2 or 3 was 5 9 10-3 M. The reaction mixtures
were kept in a water bath (50 °C) and the progress of the
reactions was followed by UV–Vis and HPLC at the 0, 150,
and 300 min time points.
The saturated matrix solution was freshly prepared by
dissolving a-cyano-4-hydroxycinnamic acid in acetonitrile/
0.1 % TFA (1:1, v/v). Freshly prepared incubation mixture
(1 mm3, 300 min incubation time) was loaded onto the
MALDI target plate (MTP 384 target plate ground steel TF,
Bruker Daltonics) and was mixed with 1 mm3 of saturated
matrix solution. Each spectrum was preceded by accumu-
lating data from 500 consecutive laser shots. The method
of identification included the mass to charge ratio (m/z) from
1 to 1,000.
MALDI-TOF mass spectrometry
MALDI-TOF mass spectrometry was performed on an
Autoflex II MALDI-TOF/TOF mass spectrometer (Bruker
Daltonics, Bremen, Germany) equipped with a 337-nm
pulsed nitrogen laser (MNL-205MC; LTB Lasertechnik
Berlin, Berlin, Germany). Spectra were recorded in the
reflectron detector mode as described previously [29, 30].
The ions were accelerated under delayed extraction con-
ditions (120 ns) in positive and negative ion modes with an
acceleration voltage of 20.00 kV. External calibration was
performed in each case using a Bruker peptide calibration
standard (no. 206195). Bruker FlexControl 2.4 software
was used for controlling the instrument. Data acquisition
and spectrum evaluations were performed by means of
Bruker FlexAnalysis 2.4 software.
UV–Vis and HPLC with UV–Vis and ESI-MS detection
UV–Vis and HPLC–UV–Vis measurements
UV–Vis measurements were performed on Specord M40
(Carl Zeiss Jena, Germany) and Secomam Anthelie Light
(Secomam, France) UV–Vis spectrophotometers using
1-cm-path-length quartz cuvettes at ambient temperatures.
HPLC analyses were performed with an Agilent 1100
(Agilent Technologies, Waldbronn, Germany) integrated
HPLC system equipped with a quaternary pump, a degas-
ser, an autosampler, and a UV–Vis diode array detector.
Data were recorded and evaluated by use of Agilent Chem
Station (Rev. A.10.02) software. The compounds were
123

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P. Perjesi et al.
1112
Fig. 6 HPLC–UV and HPLC–ESI-MS chromatograms of the reac-
tion mixture of 2 with GSH after 300 min incubation time. a HPLC–
UV chromatogram (k = 212 nm); b selected ion chromatogram
(m/z = 586.182); c ESI mass spectrum (m/z = 50–2,000); d ESI mass
spectrum (m/z = 230–570)
separated on a Spherisorb ODS2 (150 9 4.6 mm i.d.,
particle size 5 lm) column (Waters, Milford, MA, USA).
Chromatography was performed at room temperature. The
flow rate was 1.2 cm3/min. Detection was UV (k = 210
and 260 nm). The injection volume was 10 mm3.
(150 mm 9 2.1 mm i.d., particle size 2.6 lm) column
(Phenomenex, Torrance, USA). The flow rate was
200 mm3/min; the injection volume was 1 mm3. Hystar 3.2
(Bruker Daltonics, Bremen, Germany) and Bruker micro-
TOF control 1.3 software were used for controlling the
instrument. Data acquisition and spectrum evaluations
were performed by means of Bruker Data analysis 4.0
software.
A binary gradient consisting of mobile phases A and B
(A: methanol–0.1 % trifluoroacetic acid; B: water–0.1 %
trifluoroacetic acid) was applied for the chromatographic
separation. The compounds were separated with the fol-
lowing gradient profile: 40 % A for 5 min followed by a
10-min linear gradient to 90 % and an isocratic period of
5 min. The column was equilibrated to the initial condi-
tions with a 2-min linear gradient to 40 % A and an
isocratic period of 5 min.
A binary gradient consisting of mobile phases A and B
(A: methanol–0.1 % formic acid; B: water–0.1 % formic
acid) was applied for the chromatographic separation. The
compounds were separated with the following gradient
profile: 40 % A for 5 min followed by 10 min linear gra-
dient to 90 % and an isocratic period of 5 min. The column
was equilibrated to the initial conditions with a 1-min
linear gradient to 40 % A and an isocratic period of 5 min.
A Bruker microTOF mass spectrometer, equipped with
an ESI source was used for mass detection. The ionization
source was operated with an endplate potential of 3.8 kV in
the positive ion mode. The following electrospray param-
eters were kept constant during the analysis: drying gas
HPLC–ESI-MS measurements
Analyses were performed with a complex Ultimate 3000
(Dionex, Sunyvale, USA) micro HPLC system equipped
with a quaternary pump, a degasser, and a Bruker micro-
TOF mass spectrometer (Bruker Daltonics, Bremen,
Germany). Separations were performed on a Kinetex C-18
123

Conjugation reaction of two cytotoxic cyclic chalcone analogues with glutathione
1113
Fig. 7 HPLC–UV and HPLC–ESI-MS chromatograms of the reac-
tion mixture of 3 with GSH after 300 min incubation time. a HPLC–
UV chromatogram (k = 212 nm); b selected ion chromatogram
(m/z = 570.227); c ESI mass spectrum m/z = 50–2,000); d ESI mass
spectrum (m/z = 230–560)
(N2) flow 8 dm3/min, drying gas temperature 210 °C,
nebulizer pressure 20 psi. Spectra were acquired in the
mass/charge ratio (m/z) range of 50–2,000.
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Acknowledgments This study was supported the Faculty of Med-
icine Research Fund (AOK-KA 34039-12/2009, AOK-KA 34039-11/
7. Dinkova-Kostova AT, Massiah MA, Bozak RE, Hicks RJ,
Talalay P (2001) Proc Natl Acad Sci 98:3404
´
2009, and AOK-KA 2011), University of Pecs, the Hungarian
8. Jin YL, Jin XY, Jin F, Sohn DH, Kim HS (2008) Arch Pharm Res
31:1145
9. Wang J, Wang S, Song D, Zhao D, Sha Y, Jiang Y, Jing Y, Cheng
M (2009) Chem Biol Drug Des 73:511
National Scientific Research Foundation (OTKA) grant no. K72592,
D048294, CNK 78480, and the TIOP 1.3.1-10/1-2010-0008 and TIOP
1.3.1-07/1 grants. The authors express their special thanks to Dr.
´
´
´
Laszlo Mark (Department of Biochemistry and Medical Chemistry,
University of Pecs) and Dr. Zsuzsanna Rozmer (Department of
10. Dimmock JR, Kandepu NM, Nazarali AJ, Kowalchuk TP, Mot-
aganahalli N, Quail JW, Mykytiuk P, Audette GF, Prasad L,
´
´
Pharmaceutical Chemistry, University of Pecs) for their kind assis-
tance in some parts of the experimental work.
´
Perjesi P, Allen TM, Santos CL, Szydlowski J, De Clercq E,
Balzarini J (1999) J Med Chem 42:1358
´
11. Dimmock JR, Zello GA, Oloo EO, Quail JW, Kraatz HB, Perjesi
P, Aradi F, Takacs-Novak K, Allen TM, Santos CL, Balzarini J,
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