Synthesis, structural characterization and biological activity of novel Knoevenagel condensates on DLD-1 human colon carcinoma

Article abstract of DOI:10.1016/j.bmcl.2017.04.031

Biologically active Knoevenagel condensates (1–14) of diarylheptanoids: 1,7-bis(3-methoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione and 1,7-bis(3-ethoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione, were synthesized and structurally characterized. Compounds 1–14 exhibited cytotoxicity against colon carcinoma cells, and their antiproliferative effect was associated with a significant decrease of multidrug resistance proteins. One of the underlying mechanisms of these effects is the reduction of intracellular and extracellular SOD enzymes by compounds 1, 12 and 14, which render the tumor cells more vulnerable to oxidative stress.

Full text of DOI:10.1016/j.bmcl.2017.04.031

Accepted Manuscript
Synthesis, structural characterization and biological activity of novel Knoeve-
nagel condensates on DLD-1 human colon carcinoma
Lucia Rišiaňová, Eva Fischer-Fodor, Jindra Valentová, Corina Tatomir,
Nicoleta Corina Decea, Piroska Virag, Iveta Pechová, Ferdinand Devínsky,
Natalia Miklašová
PII:
S0960-894X(17)30409-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.031
BMCL 24884
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 February 2017
10 April 2017
11 April 2017
Please cite this article as: Rišiaňová, L., Fischer-Fodor, E., Valentová, J., Tatomir, C., Corina Decea, N., Virag, P.,
Pechová, I., Devínsky, F., Miklašová, N., Synthesis, structural characterization and biological activity of novel
Knoevenagel condensates on DLD-1 human colon carcinoma, Bioorganic & Medicinal Chemistry Letters (2017),
doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.031
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Synthesis, structural characterization and biological activity of novel Knoevenagel
condensates on DLD-1 human colon carcinoma
Lucia Rišiaňová a, Eva Fischer-Fodor b, c, Jindra Valentová a, Corina Tatomir b, Nicoleta
Corina Decea d, Piroska Virag b, Iveta Pechová a, Ferdinand Devínsky a, Natalia Miklašová a*
a
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University in Bratislava,
Kaliničiakova 8, Bratislava 83232, Slovakia
b
Institute of Oncology "I. Chiricuta" Cluj-Napoca, 400015, Romania,
c
Medfuture Research Center, University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj Napoca, 400012,
Cluj-Napoca, Romania
d Department of Physiology, University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj Napoca, 400012,
Cluj-Napoca, Romania
*Corresponding author
Abbreviations
ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
DPPH: di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium
MDR1: multidrug resistance gene 1
MRP1: multidrug resistance-associated protein 1
PBS: Phospate–buffered saline
SOD: superoxide dismutase
GSH: glutathione
GSSG: glutathione disulfide
Abstract
Biologically active Knoevenagel condensates (1-14) of diarylheptanoids: 1,7-bis(3-methoxy-
4-hydroxyphenyl)hepta-1,7-diene-3,5-dione and 1,7-bis(3-ethoxy-4-hydroxyphenyl)hepta-
1,7-diene-3,5-dione, were synthesized and structurally characterized. Compounds 1-14
exhibited cytotoxicity against colon carcinoma cells, and their antiproliferative effect was

associated with a significant decrease of multidrug resistance proteins. One of the underlying
mechanisms of these effects is the reduction of intracellular and extracellular SOD enzymes
by compounds 1, 12 and 14, which render the tumor cells more vulnerable to oxidative stress.
Keywords: Knoevenagel condensates; curcuminoids; antioxidant activity; cytotoxicity; SOD-
mimetic activity; multidrug resistance
Curcumin, a remarkable pharmacologically active product, exhibits low oral
bioavailability a rapid first-pass metabolism and excretory system.1 Therefore, new
structurally modified derivatives of curcumin with better pharmacokinetic attributes are
required. An affordable alternative are Knoevenagel condensates of curcumin, synthesized by
reacting the curcuminoids with appropriate aldehydes. The products of condensations are non-
enolisable compounds.2 Previous studies have shown that Knoevenagel condensates of
curcumin possess higher antibacterial activity than curcumin itself. Substituted benzylidene
rings with electron releasing groups in the 2nd and 4th positions demonstrated extensive
activity against S. aureus and P. aeruginosa.3 Moreover, 4-arylidene curcumin analogues
proved to have antiproliferative activity.4,5 Several derivatives with various substituted
benzylidene rings have showed significantly higher antitumor activity against 5 tested cancer
cell lines (A549, HepG2, MCF-7, SW480 and CNE2) than their curcumin analogues.6
Knoevenagel condensates are capable of complexation with metal ions through keto-groups.2
Copper complexes of Knoevenagel condensates are more cytotoxic on H1299 and KBM-5
cell lines than curcumin alone.7
The present work is focused on the synthesis, structural characterization (NMR, IR,
UV, HR-MS spectra) and biomedical activity of Knoevenagel condensates derived from 1,7-
bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione and 1,7-bis(4-hydroxy-3-
ethoxyphenyl)hepta-1,6-diene-3,5-dione, respectively. Compounds 1-14 were the subject of
analytical ABTS and DPPH assays used to evaluate antioxidant capacity. All condensates
were investigated in vitro on human colon carcinoma cell lines DLD-1, for the determination
of cytotoxicity, SOD-mimetic activity (SOD1, SOD2, SOD3) and their capacity to oxidize
glutathione (GSH).
Knoevenagel condensates were synthesized using a previously reported general
procedure6 (Scheme 1). The aromatic aldehyde (2 equiv.) and catalytic amounts of piperidine
and acetic acid were added to a solution of a pure arylheptanoid (1 equiv.) in toluene. The
reaction mixture was stirred overnight at 140 °C and the final product was isolated by column
chromatography (ethyl acetate:hexane, 2:1). Formerly reported condensates 1, 2, 4, 5, 7 2,8,9

were obtained as yellow-orange powders with satisfactory yields (39-75%). The new
derivatives 3, 6 and 8-14, were isolated as orange powders in relatively good yields (11-75%).
(Supplementary information).
O
OH
O
R
R
+
R1
HO
OH
R2
140 o
C
piperidine, acetic acid
toluene
(overnight)
O
O
R
R
HO
OH
1
2
1
2
1 R=OCH R =OCH R =OH
8 R=OCH CH R =OCH R =OH
3,
3,
2
3,
3,
1
2
1
2
2 R=OCH R =R = OCH
9 R= OCH CH R =R = OCH
2 3, 3
R2
3,
3
1
2
1
2
3 R=OCH R =OCH CH R =OH
10 R=R = OCH CH R =OH
2 3,
3,
2
2
3,
1
1
2
R1
4 R=OCH R =H, R =OH
11 R= OCH CH R =H, R =OH
2 3,
3,
1
2
1
2
5 R=OCH R =R =OH
12 R= OCH CH R =R =OH
3,
2
2
3,
1-14
1
2
1
6 R=OCH R =R = OCH CH
13 R=R =R = OCH CH
3,
2
3
2
1
3
2
1
2
7 R=OCH R =R =H
14 R= OCH CH R =R =H
2 3,
3,
Scheme 1. Synthesis of Knoevenagel condensates 1-14
The antioxidant activity of Knoevenagel condensates 1-14 was determined in terms of
radical scavenging activity using the stable radical DPPH. Condensates 1-14 and standards
1,7-bis(3-methoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione (C1) and 1,7-bis(3-ethoxy-4-
hydroxyphenyl)hepta-1,7-diene-3,5-dione (C2) were tested as solutions in DMSO at
a concentration of 1 mM. The DPPH assay shows that compound 5 has the highest activity
among the fourteen synthesized conjugates and slightly higher antioxidant activity compared
to the starting curcumin C1. When the antioxidant activity was followed by ABTS assay,
compounds 1, 5, 8, 12 exhibited higher activities compared to the other conjugates, C1 and
C2, respectively. (Figure 1, Table S1-Supplementary information). These results follow
the theory where the OH phenolic groups increase antioxidant activity. Based on the DPPH
and ABTS values, the cytotoxicity of compounds 1-14 was determined in terms of a half
maximal inhibitory concentration IC50, (Table S2, Figure S1- Supplementary information)
calculated from the plotted graph of scavenging activity against the concentration of tested
compounds.

Figure 1. Inhibition of ABTS at 100 µM and inhibition of DPPH at 1 mM for compounds 1-
14 and standard arylheptanoids C1 and C2
The synthetic curcuminoids are known as compounds which are capable of
modulating cell response to oxidative stress and the drug resistance mechanisms.10 While the
antioxidant activity of several curcumin structures in normal human cells is protective, in
tumor cells it might be beneficial in the inhibition of antioxidant enzymes. Using the
Knoevenagel condensation, we synthesized antiproliferative derivatives which target the
redox signaling through the antioxidant enzymes. Knoevenagel condensates 1-14 have the
capacity to inhibit DLD-1 colon cancer cells growth; their cytotoxicity was quantifieda using
the half maximal inhibitory concentration (IC50), which is below 10 µM for 1, 2, 3, 4, 6, 7, 8,
10, 12, 13 and 14. With three exceptions (5, 9 and 11) the Knoevenagel condensates
cytotoxicity was higher than the standard platinum-based drug oxaliplatin. Compounds 5, 9
and 11 displayed IC50 values under 100 µM; however, they are significantly higher in the
whole series (Table 1).

Table 1. The Knoevenagel condensates influence on cell growth inhibition and cell
proliferation: half maximal inhibitory concentrations (IC50) of compounds 1-14 calculated
from the nonlinear dose-response curves and the capacity of Knoevenagel condensates 1-14 to
diminish the living cells reducing potential
Cytotoxicity
Cell proliferation
Deviation
from 0
SD Log
IC50
IC50 [µM] best
Hill slope
P value
0.002
0.015
0.017
0.001
0.032
0.007
0.014
0.081
0.022
0.011
0.158
0.003
0.006
0.091
0.049
0.009
Compound
a
fit value
---------
5.063
9600 ±1400
-990 ± 240
-960 ± 240
-1000 ± 100
-387 ± 72
S
S
Untreated cells
----------
0.09
1
0.18
0.12
0.10
0.17
0.11
0.20
0.16
0.24
0.60
0.18
0.11
0.18
0.12
0.20
S
3.073
2
S
4.260
3
S
4.079
4
-1000 ± 200
-860 ± 210
1700 ± 720
-900 ± 250
-660 ± 150
1300 ± 750
-1700 ± 270
-430 ± 80
S
17.590
4.116
5
S
6
NS
S
5.450
7
9.634
8
S
42.041
1.360
9
NS
S
10
20.284
5.101
11
12
S
2300 ± 490
NS
S
3.454
13
-820 ± 290
-740±110
5.064
14
S
14.771
Oxaliplatin
a standard deviation was calculated in the 95% confidence interval
S - Significant
NS - Not significant
The proliferation of the cells was measured quantitatively with fluorescence methodsb at 4
hours, 24 hours, and 48 hours, by exposing the DLD-1 cells to synthesized compounds 1-14.
As reference, we used the untreated cells which exhibit a high proliferation rate, as expected.
(Table 1) The statistical significance of the changes in cell proliferation was quantified using
the Hill slope deviation from 0, in the 95% confidence interval. Condensates 1-14 have the

capacity to negatively influence cells proliferation, as proved by the decrease of the cells
number (negative hill slope) in most cases. The capacity of the Knoevenagel condensates to
inhibit the cellular cytosolic SOD1 (Cu/Zn-SOD) and the mitochondrial SOD2 (Mn- and Fe-
SOD) was also determinedc. In lysates and supernates the protein concentration was evaluated
using the Bradford method, as described earlier.11 The sum of the two isozymes reflects the
total SOD activity inside the treated or reference, untreated cells (Figures 2, 3, 4).
Figure 2. Differences between the degree of intracellular SOD1 and SOD2 superoxide
dismutases inhibition in cancer cells treated with 1-14.
Figure 3. The capacity of condensates 1-14 to inhibit SOD2 in colon cancer cells, compared
with untreated (control, C) cells

The activity of intracellular cytosolic Cu/Zn-SOD or SOD 1 and the mitochondrial
Mn-SOD or SOD2 isozymes in DLD-1 tumor cells is modulated by the treatment with 1-14
(Figure 2). The untreated DLD-1 cells cytoplasm is prone to activate the intracellular SOD,
an expected phenomenon in mutant tumor cells. The SOD1+SOD2 isozymes are significantly
inhibited by compounds 1-5, 9, 12 and 14, all variances being statistically significant (one-
way ANOVA, Dunnet post-test, in a 95 % confidence interval). Thus, the DLD-1 tumor cells
capacity to respond to the oxidative stress mediated by the superoxide ion is diminished, and
so, the viability of the tumor cell population is reduced. However, compounds 6, 7, 10, 11 and
13 do not inhibit intracellular SOD activity; the negative values (-4.9 to -18.72) show a better
SOD activity which can protect the tumor cell from the desirable loss of viability through the
action of Knoevenagel derivatives (Figure 3). SOD2, measured separately by inactivating
SOD1 with 2 mM sodium cyanate in the cell lysates, proved to be less affected, the variations
of the percent of O2- inhibition being not more than -2.89 to +3.17%. As stated by the one-
way ANOVA test (p<0.05), compounds 1, 2, 6, 9, and 14 significantly inhibit SOD2, while
treatment with 11 generates more active mitochondrial Mn-SOD. Overall, the influence of the
tested compounds on intracellular SOD enzyme modulation was more important for SOD1
than for SOD2, which has a lower contribution to the percentage of inhibition.
Figure 4. The capacity of Knoevenagel condensates 1-14 to modulate SOD3, the extracellular
Cu/Zn-SOD superoxide dismutase activity
Considering that in the untreated DLD-1 cells supernates display a basal value of
37.36% inhibition of SOD3, only treatment with 12 inhibited the extracellular SOD, whereas

treatment with 2, 3, 8, 9 and 10 caused an increase of this enzyme activity (one-way ANOVA,
p<0.05), which is reflected in the cells growth environment (Figure 4). Comparing the
intracellular and the extracellular isozymes, 1, 12 and 14 display a tendency to reduce SOD
activity, exposing the DLD-1 k-ras mutant tumor cells to oxidative stress mediated cell death.
The action of 2, 3, 4, 5, 8 and 9 on SODs was divergent; it was dissimilarity between the
down regulatory effect on cytosolic Cu/Zn-SOD (SOD1) and the upregulation of extracellular
Cu/Zn-SOD (SOD3). Only treatment with 6, 7, 10, 11 and 13 leads to the convergent
activation of SOD1 and SOD3 isozymes following treatment inside the cells and in the
extracellular environment. Compounds 10 and 13 do not reduce MnSOD (SOD2) activity,
therefore, despite their cytotoxicity, it is very likely an activation of the DLD-1 tumor cells
survival pathway through redox regulation.
The expression of MDR1 and MRP1, biomarkers of multidrug resistance, was
measuredd using a fluorometric method, as described previously.12 The basal value of MRP1
expression was relatively high in resistant, k-ras mutant DLD-1 cells, in accordance with
earlier data.13 As a result of exposure of DLD-1 cells to 1-4, 6, 7, 8, 11-14, a significant
decrease in multidrug resistance proteins was observed (Figure 5). The drug efflux from the
cell was diminished. This generates a higher cellular uptake, and the cells became more
susceptible to lose their capacity to proliferate.
a.
b.
Figure 5. a. The effect on the multidrug resistance protein MRP1 (ABCC1) mediated drug
transport in DLD-1 colon tumor cells for a 24-hours exposure to 1-14; the untreated cells are
represented in column C. b. Electrophilicity of compounds 1-14; the rate constant of reaction
kGSH is an indicator of the reaction between GSH and the test compounds and suggests the GSH
degradation via electrophilic attack by the test molecules.

Consistent with the SOD inhibition, compounds 1, 12 and 14 can counteract the drug
resistance mechanisms as well, but we found no correlation between multidrug resistance
decrease and superoxide dismutase inhibition (two-tailed Pearson correlation, p>0.05).
The glutathione (GSH) have been used as a model nucleophile to test the condensates
1-14 electrophilic charactere, following an earlier described method.14 The chemical
reactivity, in terms of kGSH, which represent a total rate constant, reflects the total GSH loss
due to addition of the electrophiles. The studied Knoevenagel condensates present different
reactivities, 1 and 4 have a certain electrophilicity, but most of the molecules (2, 3 and 6-14)
exhibit a low electrophilic character in comparison with several references (eg. ethyl
propiolat, kGSH = 105 ± 5 mol/L/min, n-butyl acrylate, kGSH = 8.54 ± 0.20 mol/L/min or 3-
methyl-3-pentene-2-one, kGSH = 0.779 ± 0.018 mol/L/min), and 5 gave no reaction with GSH.
The in vitro effect of 1-14 against the intracellular glutathione 15 and the capacity of
condensates to modify the ration between the reduced GSH and its oxidized form, glutathione
disulfide (GSSH), was also quantified using a fluorescence methodf. In tumor cells, the
GSH/GSSG ratio tends to be modified as compare with the normal cells, and the decrease of
GSH, in the favour of GSSG, is an indicator of programmed cell death.16 GSH was augmented
by all compounds, except of 13, while in GSSG was observed a reduction, except for
compound 9 (Table 2).
Table 2. The glutathione level in the tumor cells treated with Knoevenagel condensates: the
reduced glutathione (GSH), the oxidized glutathione (GSSH) and the proportion between
reduced and oxidized forms (GSH/GSSH).
Compound
GSH
GSSG
GSH/ GSSG
(nmol/mg
protein)
(nmol/mg
protein)
(nmol/mg
protein)
Untreated
cells
2.19 ±0.18
4.56±0.06
3.93±0.26
5.12±0.01
4.80±0.17
2.60±0.13
0.69±0.00
2.80±0.02
4.21±0.04
1.02±0.01
1.20±0.13
0.50±0.06
3.19±0.20
1.63±0.28
0.93±0.04
5.00±0.09
3.99±0.19
5.17±0.65
1
2
3
4
5

4.27±0.75
4.47±0.09
9.75±0.45
3.24±0.16
3.20±0.33
3.59±0.00
4.41±0.09
2.12±0.14
3.27±0.03
1.88±0.17
1.40±0.00
4.30±0.23
0.49±0.08
0.84±0.01
3.13±0.33
1.16±0.07
3.24±0.00
0.48±0.05
2.28±0.06
3.19±0.22
2.27±0.06
6.59±0.25
3.80±0.03
1.15±0.01
3.78±0.02
0.65±0.01
6.89±0.12
6
7
8
9
10
11
12
13
14
The kGSH of 1-14 measured outside the biologic environment, is well correlated with
the GSH and GSSG changes inside the treated cell (two-tailed nonparametric Pearson
correlation, p<0.05). The increase of DLD-1 multidrug resistance is inversely proportional
with the electrophilic character of 1-14 (p=0.0058, very significant) and with the intracellular
GSH decrease (p=0.019, significant). Nevertheless, kGSH, intracellular GSH and GSSG are not
correlated to SOD, cytotoxicity or cell proliferation. Compounds 1-6, 8, 9, 11, 12 and 14 can
inhibit the proliferation of DLD-1 cells in vitro.
Cytotoxicity of these compounds leads to a reduction of multidrug resistance proteins
expression inside the cells treated with 1-4, 6-8 and 11-14, which correlates well with their
antiproliferative effect. Furthermore, the antiproliferative activity was associated with the
inhibition of the intracellular antioxidant enzymes SOD1 and SOD2 by 1-5, 9, 12 and 14. The
extracellular environment was also influenced through SOD3 inhibition when treatment with
1, 12 and 14 causes vulnerability of the colon carcinoma cells to the oxidative stress. The ratio
of oxidized glutathione inside the treated cell is strongly diminished in 1, 2, 11 and 13,
suggesting the DLD-1 cell transition towards programmed cell death. The biologic outcome
of 1-14 against tumor cells provides certain potential towards their employment as tumor
growth inhibitors.
Acknowledgements
This work was supported by the Slovak Research and Development Agency under the
contract No. APVV-0516-12 and by VEGA 1/0346/16. This publication utilizes the research

results of the CEBV project, ITMS 26240120034. Special thanks are also addressed to Vlad
Alexandru Toma from National Institute for Research and Development of Isotopic and
Molecular Technologies, Cluj-Napoca, Romania.
Supplementary information
References and notes
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2007;37:19-27.
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7 Zambre AP, Kulkarni VM, Padhye S, et al. Bioorg. Med. Chem. 2006;14:7196-7204.
8 Youssoufi MY, Sahu PK, Agarwal DD, et al. Med Chem Res. 2015;24:2381-2392.
9 Zheng XH, Shao YX, Li Z, et al. J Sep Sci. 2012;35:505-512.
10 Lopes-Rodrigues V, Sousa E, Vasconcelos MH. Pharmaceuticals (Basel). 2016;9:71.
a The in vitro studies were performed on DLD-1 human colon carcinoma cell cultures
acquired from European Collection of Authenticated Cell Cultures (ECACC, Salisbury,
United Kingdom). DLD-1 cells were grown in RPMI-1640 cell culture media supplemented
with fetal bovine serum, glutamine and penicillin-streptomycin solution (all media and
supplements from Sigma Aldrich). Cells were seeded into 96-well microplates, about 20x103
cells/well in 190 μL media, and on 6-well plates, respectively, 3x105 cells in 2.85 mL media
(Nunclon delta surface plates from Thermo Fischer Scientific, Waltham, MA, USA). After
24-hours of incubation in humidified atmosphere with 5% CO2 the cells were attached to the
surface and the wells were treated with the solutions containing Knoevenagel condensates 1-
14. The treatment was made using a quantity of 10 μL solution/well in 96-well plates, and 150
μL in 6-well plates, respectively. Laboratory instrumentation: Class II laminary hoods Lamil
Plus 13 from Karstulan Metalli Oy, Karstula, Finland; 32R centrifuge with spin-out rotor from
Hettich Lab Technology, Tuttlingen, Germany; Observer D.1 inverted phase fluorescence
microscope from Zeiss (Jena, Germany. The cytotoxicity of the compounds was measured
using the MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, from
Sigma Aldrich). The formazan crystals were solubilized in DMSO (from Titolchimica, Italy),
and the 96-well plates were measured in colorimetry at 570 nm wavelength using a Synergy
2.0 microplate reader (from BioTek Company, Winooski, USA). Three independent
measurements were performed for every compound. The control values were the untreated
cells; a concentration range of 1-100 µM (final concentrations in cell culture media) was used
for all condensates.
b For quantitatively proliferation testing, the DLD-1 cells were seeded on 96-well black Costar
assay plates with clear bottom (from Corning Incorporated, Corning, NY, USA), at a 3x104
cell/well density in 190 µL media. After 24-hours incubation the adhered cells were treated
similar as for the cytotoxicity test; Alamar Blue Cell Viability Reagent (from Life
Technologies Corporation, Carlsbad, USA), was added to each well and fluorescence

monitoring was at 620 nm wavelength (excitation at 540 nm). For the proliferation testing, 10
µL of 100 µM Knoevenagel condensates were used for each well; all measurements were
made in triplicate.
c The enzymatic activity was measured using the Superoxid Dismutase Assay kit (Trevigen
Incorporation, Gaithersburg, MD, USA), following the manufacturer’s instructions. A number
of 5x 105 cells were seeded on 6-well plates, and were treated with 10 µL of 100 μM
compounds 1-14; as reference, untreated cells were used. After 24 hours, the cell culture
media was harvested from all wells; the plates were subjected to cell lysis using an NP-40 cell
lysis buffer (Abcam, Cambridge, UK). The SOD reaction buffer (provided by the
manufacturer), xantine solution and deionized water (1500 μL), then NBT solution and
samples (lysates or cell culture media) were added to a cuvette. Measurements were made at
550 nm using a V-530 spectrophotometer (Jasco, USA). After adding xantine oxidase, the
samples were measured after 30 seconds and after 5 minutes and the rate of increase in
absorbance units (A) per minute was determined using the formula described in
Supplementary Material.
d The DLD-1 cells were plated on 96-well plates and exposed for 24 hours to 10 µL/well of
100 μM 1-14, They were then treated with sensor dye provided by the Ab112142 MDR assay
kit (Abcam, Cambridge, UK), and after one hour of incubation the measurements were
performed using the Synergy 2 platform at 485 nm emission with a 620 nm absorption
wavelength.
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e Reduced GSH, DMSO, dithionitrobenzoic acid, disodium hydrogen phosphate x 12 H2O and
potassium dihydrogen phosphate (anhydrous) were obtained from Sigma-Aldrich, all of
analytical purity. Reaction rate constants were determined using an UV-vis spectrophotometer
at 412 nm (VWR 1600-PC Spectrophotometer). The rate constants of reaction between the
GSH and the electrophilic test compounds, kGSH were determined by the method of Böhme et
al., employing first-order kinetics for less reactive molecules and second-order kinetics for the
more reactive electrophiles.
14 Böhme A, Thaens D, Paschke A, Schüürmann G. Chem ResToxicol. 2009; 22:742-750.
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Embryol. 2013; 54:1097-106.
f. The treated/untreated cells were lysed as for SOD method, the protein was precipitated with
10% trichloroacetic acid and re-suspended in pH 8 phosphate buffered saline solution (all
reagents from Sigma Aldrich). GSH were determined using the fluorescence probe ortho-
phthalaldehyde; the measurements were made at 350 nm/420 nm using an LS 45 fluorescence
spectrometer (from Perkin Elmer, Waltham, MA, USA). GSSG was precipitated with
metaphosphoric acid 10%, then mixed with N- ethylmaleimide, subjected to ortho-
phthalaldehyde and measured, like previously described for GSH. The quantitative evaluation
was made using a standard curve.
16 Circu ML, Aw TY. Biochim Biophys Acta. 2012; 1823:1767–1777.

45
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10
5
0
Cytotoxicity of 1-14