Ectonucleotidase Inhibitory and Redox Activity of Imidazole-Based Organic Salts and Ionic Liquids

Article abstract of DOI:10.1002/cmdc.201800520

Cytotoxicity against cancer and normal cells, inhibition of ectonucleotidase, and redox properties of a new group of imidazole-based organic salts and ionic liquids were studied. The tetrachloroferrate salt of a 1-methylimidazole derivative of salicylic aldehyde had most prominent inhibitory activity against ectonucleotidase as well as a higher cytotoxicity against HeLa cells and lower cytotoxicity against BHK-21 cells than the reference compound carboplatin. The studied compounds exhibited a moderate level of antioxidant activity with better results for the salicylic aldehyde derivatives than for spiropyrans. Moreover, these compounds did not generate singlet oxygen.

Full text of DOI:10.1002/cmdc.201800520

Accepted Article
Title: Ectonucleotidase inhibitory and redox activity of imidazole-based
organic salts and ionic liquids
Authors: Fliur Macaev, Veaceslav Boldescu, Natalia Sucman, Sidra
Hassan, Jamshed Iqbal, Mariana Neamꢀu, Joanna Lecka,
Jean Sevigny, and Denis Prodius
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To be cited as: ChemMedChem 10.1002/cmdc.201800520
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Ectonucleotidase inhibitory and redox activity of imidazole-based
organic salts and ionic liquids
Veaceslav Boldescu,[a] Natalia Sucman,[a] Sidra Hassan,[b] Jamshed Iqbal,[b] Mariana Neamtu,[c] Joanna
Lecka,[d] Jean Sévigny,[d] Denis Prodius,[e] and Fliur Macaev*[a]
[a]
Dr. Veaceslav Boldescu, Dr. Natalia Sucman, Prof. Fliur Macaev
Laboratory of Organic Synthesis and Biopharmaceuticals
Institute of Chemistry
3 Academiei str., 2028 Chisinau, Moldova
E-mail: flmacaev@cc.acad.md
[b]
[c]
[d]
[e]
Dr. Jamshed Iqbal, Sidra Hasan
Department of Pharmaceutical Sciences
COMSATS Institute of Information Technology
22060 Abbottabad, Pakistan
Dr. Mariana Neamtu
Interdisciplinary Research Department
„Alexandru Ioan Cuza” University
54 Lascar Catargi str., 700107 Iasi, Romania
Joanna Lecka, Dr. Jean Sévigny
Département de microbiologie-infectiologie et d'immunologie,
Faculté de Médecine, Université Laval, Québec, QC, G1V 0A6, Canada
Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
Dr. Denis Prodius
Laboratory of Bioinorganic Chemistry and Nanocomposites
Institute of Chemistry
3 Academiei str., 2028 Chisinau, Moldova
Current address: Ames Laboratory
U.S. Department of Energy and Critical Materials Institute
Ames, Iowa, 50011-3020, USA
Supporting information for this article is given via a link at the end of the document.
Abstract: Cytotoxicity against cancerous and normal cells, inhibition
of ectonucleotidase, and redox properties of a new group of
imidazole-based organic salts and ionic liquids have been studied.
The tetrachloroferrate salt of a 1-methylimidazole derivative of
salicylic aldehyde has been shown to have most prominent inhibitory
activity against ectonucleotidase, as well as a higher cytotoxicity
against HeLa cells than that of the reference compound carboplatin,
while having lower cytotoxicity towards BHK-21 cells. The studied
compounds have shown a moderate level of antioxidant activity with
better results for the salicylic aldehyde derivatives than for the
spiropyrans. Moreover, the compounds did not generate singlet
oxygen formation.
studied cytotoxicity of a group of ionic liquids and their
precursors in HeLa cells and determined increase in reactive
oxygen species production (ROS) and a consequent reduction
of mitochondrial membrane potential. At the same time,
photoinduced electron transfer has been demonstrated for 2,4,6-
triphenylthiapyrylium in ionic liquid bmim-PF6.[21] This study has
demonstrated that a highly polar medium formed by ionic liquid
stabilizes the formed organic intermediates, thus increasing their
live span. A thorough review of the biological effects of OSILs
and particularly of their anticancer effect has detected three
main mechanisms of their antiproliferative activity: oxidative
stress, DNA damage and induction of apoptosis in cells.[22,23]
Anticarcinogenic properties of anthocyanidins are attributed to
their property to scavenge different mutagenic types of free
radicals like reactive oxygen and nitrogen species(ROS/RNS).[24]
At the same time, despite their antioxidative nature,
anthocyanidins have been found to selectively induce a strong
prooxidative and proapoptotic effect in tumour cells.[25] While it is
well known that intracellular generation of singlet oxygen equally
induces cell death in both cancerous and noncancerous cells,
extracellular singlet oxygen has a more selective action on
tumour cells via membrane-associated catalase inhibition and
reactivation of intracellular ROS/RNS-dependent apoptosis-
inducing signalling, while having no effect on non-malignant
cells.[26]
Introduction
The multifunctionality including the anticancer potential of
organic salts and ionic liquids (OSILs) has been widely
discussed recently.[1-11] Their attractiveness for pharmaceutical
production is attributed to such advantageous properties as
simplicity in preparation and purification, low costs, tunable
permeability through biological barriers, etc.[12]
Most of the previous studies by other groups[13–17] on OSILs with
more or less selective cytotoxicity against different lines of
cancerous cells have mainly demonstrated the antiproliferative
activity. Other works made attempts for structure-cytotoxic
activity relationship studies that demonstrated higher toxicity for
OSILs containing cations with longer chains and lower toxicity
for compounds with functionalized side chains in cations as
compared to non-functionalized ones.[14,18,19] Wang et al.[20]
Here, we report a group of imidazole-based OSILs 1 – 8 (Figure
1) with cytotoxic activity comparable with that of carboplatin
against cancerous cells and much lower against noncancerous
cells. Biological tests of the obtained compounds have
demonstrated that they possess inhibitory activity against two
ectonucleotidase isozymes and this could be one of their main
1
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antiproliferation mechanisms of action. The results of the
exploration of the singlet oxygen formation and antioxidant
activity of the studied compounds are also presented.
same time, the C-O-C stretching vibration observed at 1262 cm-1
and 1076 cm-1. Moreover, the C-Ospiro stretching frequencies
were noticed at 983 cm-1 - 906 cm-1 that point to the
indolinobenzospiropyran structure. The C=O bond vibration that
appears only in open state of spiropyrans is not appearing in the
IR spectra of 5-8 in the region 1720-1725 cm-1.
N
N
O
HO
N
N
Cl
N O
Cl
Cl Fe Cl
Cl
Cl Fe Cl
Cl
5
6
7
1
2
3
X-ray investigation of crystal structure (4)
N
N
O
N
The X-ray measurements for 4 were carried out on a Stoe IPDS
II area detector diffractometer at room temperature, using
graphite monochromated Mo-Kα radiation (0.7107 Å).
Compound 4 crystallizes in the monoclinic P21/n space group
(see Experimental Section/Chemistry) and its asymmetric unit
contains a [FeCl4]- unit and one protonated ligand molecule for
charge balance purposes (Figure 2).
N
Cl
Cl
N O
HO
Cl Fe Cl
Cl
Cl Fe Cl
Cl
N
N
O
N
N
Cl
HO
Cl
N O
Cl Fe Cl
Cl
Cl Fe Cl
Cl
N
N
O
N
N
Cl
Cl
HO
N O
Cl Fe Cl
Cl
Cl Fe Cl
Cl
4
8
Figure 1. Imidazole-based OSILs 1-8.
Results and Discussion
Chemistry
The synthesis of the compounds 1-8 has been performed
according to the Scheme 1.
R
N
-
FeCl4
O
N
HO
R = -CH3, -C2H5, -CH=CH2, -C4H9
1 - 4
R
FeCl3*6H2O
R
N
N
Figure 2. Molecular fragment of compound 4.
O
O
O
-
Cl
Cl
N
HO(CH2O)nH
HCl conc.
N
HO
HO
HO
The iron(III) atom is situated in the center of an ideal tetrahedron.
The stability of this architecture is further supported by the
formation of several weak interactions including C-H···O and C-
H···Cl bonds between atoms from two different ligands as well
weak π-π interactions between adjacent salicylic units.
H3C
CH3
5-chloromethylsalicylic
aldehyde
salicylic
aldehyde
CH2
N
CH3
R
R
H3C
N
CH3
O
H3C
CH3
N
-
N
FeCl4
FeCl3*6H2O
Ecto-5'-Nucleotidase Inhibition Studies and SAR
-
Cl
N
N
N
Imidazolium-bearing OSILs 1-8 have been synthesized and
tested against two isozymes of ecto-5'-nucleotidase i.e. h-e5'NT
and r-e5'NT. Most of the compounds from both series exhibited
maximum inhibitory potential towards both isozymes but few
derivatives from either series exhibited selective inhibition
towards human isozymes (Table 1). Among all imidazole
derivatives compound 4 was found as the potent inhibitor. This
compound exhibited non-selective and almost equipotent
behaviour towards both isozymes i.e. h-e5'NT and r-e5'NT with
O
CH3
CH3
5 - 8
5' - 8'
Scheme 1. Synthesis of imidazole-based OSILs 1-8.
A detailed description of each step is offered in the Experimental
section. The FT-IR spectra of the obtained tetracholoroferrates
5-8 include an approximate stretching ranging from 3200 to
2800 cm-1 corresponding to the C-H stretching vibration. At the
2
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an IC50 value of 1.14±0.05 and 1.93±0.21 µM, respectively. It
can be suggested that the substitution of methyl group might be
responsible for its maximum inhibition towards both h-e5'NT and
r-e5'NT. Although this compound was found as potent one but
the aim of selectivity was achieved in case of 5, 6 and 7 as they
exhibited selective inhibition towards h-e5'NT with IC50 values of
1.36±0.11, 26.1±1.68 and 4.82±0.23 µM, respectively. It was
observed from their detailed structure that the increase in
carbon number results in the reduced inhibitory activity as
observed in case of 6. The structural activity of 6 and 7
suggested that the presence of unsaturated substituent in 7
results in the 5 folds reduced activity in comparison to 6. An
inverse behaviour was observed in the case of r-e5'NT isozyme,
derivative 2, 3, and 4 exhibited almost equipotent behaviour. It
can be suggested from their structure that the presence of
instauration is favourable towards inhibition of r-e5'NT as seen
in case of 2 (1.11±0.05 µM). Compound 3 (1.23±0.01 µM)
having ethyl fragment exhibited a little bit higher inhibition as
compared to compound 4 (1.93±0.21 µM).
approximately 2 fold higher as that of positive control used i.e.
carboplatin (5.13±0.45 µM) at the same concentration i.e. 100
µM. These compounds were found safe and did not exhibit ≥10
inhibition of normal cells (Figure 3).
Growth reduction
100
(cytotoxicity) / %
90
80
70
60
50
40
30
20
10
0
2
3
4
5
6
7
8
c
Compounds
100
90
80
70
60
50
40
30
20
10
0
Growth reduction
(cytotoxicity) / %
Table 1. Ecto-5’-Nucleotidase (h-e5'NT and r-e5'NT) inhibition data for the
imidazole compounds
Compound
h-e5’NT
r-e5’NT
(IC50 (µM) ± SEM)[a] / (% inhibition ± SEM)[b]
NT
NT
1
8.14 ± 1.21a
20.2 ± 1.45a
1.14 ± 0.05b
1.36 ± 0.11a
26.1 ± 1.68a
4.82 ± 0.23a
16.1 ± 1.35a
42.1 ± 7.80a
1.23 ± 0.01a
1.11 ± 0.05a
1.93 ± 0.21b
32.9 ± 0.45b
12.7 ± 0.18b
15.8 ± 0.11b
18.1 ± 0.98a
77.3 ± 7.00a
2
3
4
5
6
7
8
c
2
Compounds
Figure 3. Cytotoxic potential of imidazolium derivatives on HeLa (up) and
BHK-21 cells (down). carboplatin as control. The mean of three
experiments ± S.D. is shown.
3
c
–
4
5
Cell Cycle Analysis and Apoptosis by Flow Cytometry
6
Compound 4 induced maximum apoptosis against HeLa cancer
cells, i.e. 30.2% as compared to respective positive control i.e.
carboplatin (24.5%). The cell cycle analysis of 4 showed that in
comparison to the negative and positive control, it intercalated
with the DNA molecule of HeLa cells and resulted in significant
apoptotic cell death with respect to S phase. Figure 4 represents
the percentages of different cell cycle phases such as G0, G1, S,
and G2/ M.
7
8
Sulfamic acid
[a] The IC50 is the concentration of compound at which 50% of the enzyme
activity is inhibited. [b] The % inhibition is caused by 100 µM of the tested
compounds, NT- not tested
100
90
Cell / %
G0/G1-phase
S-phase
80
70
60
50
40
30
20
10
0
G2/M-phase
Apoptosis
The anticancer potential of the selected derivatives was
determined against HeLa cells, in comparison to normal cells i.e.,
BHK-21 cells. Almost all the compounds exhibited more than
50% inhibition of HeLa cells and among those compounds 2, 5,
6, 7, and 8 exhibited 68%, 62%, 67%, 64%, and 67% inhibition,
respectively. While compound 3 exhibited 73% inhibition. The
maximum inhibition was observed in the case of 4, 87%. The
results were in correlation with the enzyme inhibition data. From
both series the compounds which were identified as the most
potent inhibitor of h-e5'NT were also found to inhibit maximum
cell growth of HeLa cells. 4 caused maximum inhibition and
further selected for the determination of IC50 value. It came out
control
carboplatin
Compounds
4
Figure 4. Negative control, carboplatin as positive control and compound 4
induced apoptosis in HeLa cells as demonstrated by flow cytometry analysis
employing PI. One way analysis of variance (ANOVA) was used for Data
analysis. The mean of three experiments ± S.D. is shown. ***p<0.0001
with
a
significant IC50 value 2.92±0.11 µM that was
3
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This fact can be explained by the presence of the salicylic
aldehyde moiety in the structure of these compounds.
The activity of compound 4 significantly correlates with the MTT
results as it induced significantly higher pro-apoptotic activity as
compared to positive control.
At the same time, there is no clear correlation between the
antioxidant activity of the compounds within the groups and their
structural particularities. Thus, the main structural difference
within both groups is the substituent at the imidazole cycle:
methyl, ethyl, vinyl, and butyl. However, there is no clear
influence of the substituent nature on the antioxidant activity of
the compounds. For example, the highest antioxidant activity
represented by lower EC50 has been determined for the vinyl
derivative 3 in the 1-4 group with salicylic aldehyde moiety and
for the methyl derivative 5 in the group 5 – 8. Moreover, the
general trend of the antioxidant activity change in the group of
derivatives 1 – 4 in comparison to ascorbic acid (AA) is the
following: AA > vinyl > methyl > butyl > ethyl. While the same
trend among the 5 – 8 derivatives and ascorbic acid is: AA >
butyl > methyl > vinyl > ethyl.
Morphological evaluation of Apoptosis
DAPI and PI fluorescent dyes were used to observe DNA
condensation which is a major sign of apoptosis (Figure 5).
HeLa cells were incubated with the most potent compound 1
(1.14 µM), to examine any sign of apoptosis. The obtained
results showed maximum nuclear and chromatin condensation
as compared to negative control.
EC50
6.00
5.00
4.00
3.00
2.00
1.00
0.00
4.88
3.04
2.61
2.35
Figure 5. Apoptosis evaluation by fluorescence microscopy: (A) Untreated
HeLa cells with DAPI staining; (B) Carboplatin with DAPI staining; (C) 1 with
DAPI staining; (D) Untreated HeLa cells with PI staining; (E) Carboplatin with
PI staining; (F) 4 with PI staining.
0.22
Antioxidant activity of the studied compounds
AA
5
8
7
6
Compounds
Antioxidant activity of the 1 – 8 was measured with application of
DPPH method and compared to that of the ascorbic acid
measured by the same method. In general, compounds 1 – 4
(Figure 6) have shown higher levels of antioxidant activity than
compounds 5 – 8 (Figure 7).
Figure 7. Antioxidant activity of the compounds 5 - 8 expressed in EC50. AA –
ascorbic acid. The mean of three experiments ± S.D. is shown.
Molecularity trends for each of the groups of the
compounds are the following: 3 (0.66) > 1 (0.33) > 4 (0.27) > 2
(0.18) and 8 (0.33) > 5 (0.24) > 7 (0.20) > 6 (0.13). Molecularity
represents the number of DPPH molecules that can be reduced
by one molecule of the substrate.
Previously, a group of imidazolium-based ionic liquids with
antioxidant properties has been reported by Cai et al.[27] The
group has reported on antioxidant properties of (BHT-1) MIMPF6
and other imidazolium derivatives detected via rotative bomb
oxidation test and thermal analysis. These properties allowed
the use of these antioxidants as an anti-wear and antioxidative
additive in PEG for steel/steel contacts.
4.00
3.00
2.00
1.00
0.00
EC50
2.83
1.95
1.59
1.09
Another group of imidazolium-bearing antioxidative ionic
liquids has been reported by Cai et al.[28] These compounds
contain
1-(1H-benzo[d]p[1,2,3]triazole-1-yl)methyl)-3-
methylimidazolium with different anions (PF6, BF6, NTf2) and
they can act as low molecular weight gelators. They have been
shown to increase resistance to oxygen attack in lubricants at
friction. No antioxidant activity parameters have been reported in
any of these investigations.
0.22
AA
3
4
1
2
Compounds
Figure 6. Antioxidant activity of the compounds 1 - 4 expressed in EC50. AA –
ascorbic acid. The mean of three experiments ± S.D. is shown.
Electron spin resonance investigation
4
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To follow the formation and quenching of the photosensitized
singlet molecular oxygen, 1O2, generated by our compounds, the
ESR was employed by using 2,2,6,6-tetramethyl-4-piperidinol
(TMP-OH) as a spin trap. According to the literature, the
diamagnetic scavenger TMP-OH reacts with 1O2, yielding a
been detected after irradiation of compounds in TEMPOL assay,
while the compounds have appeared to be moderate
antioxidants in DPPH assay. To our best knowledge, this is the
first study on OSILs antiproliferative activity with a multilateral
exploration of its mechanisms.
stable
paramagnetic
product,
4-hydroxy-2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPOL)[29] that can be easily
detected by ESR. Although other ROS can also lead to the
formation of TEMPOL,[30] the reactive scavenging of 1O2 by
TMP-OH is considered to be highly specific for 1O2.[30] Figure SI1
in Supporting information shows the ESR spectra obtained from
the samples containing TMP-OH in the presence of compounds
under UVA illumination.
Experimental Section
Chemistry
Synthesis of 5-chloromethylsalicylic aldehyde: salicylic aldehyde (10 g,
0.082 mol) has been added drop-wise at vigorous stirring
paraformaldehyde (7.5 g) in HCl (150 mL, ~30 wt. %). The mixture has
been stirred at room temperature for 24 h. The formed precipitate has
been subjected to filtration and washed with diethyl ether. η = 55%. mp:
80°C.
As Figure SI1 shows, no three equidistant and equi-intense
hyperfine lines in ESR spectrum, typical for TEMPOL,
comparing with control solution (containing only TMP-OH)
exposed to UVA illumination for 60 minutes, couldn’t observe.
This suggests that the compounds don’t generate singlet oxygen,
even at exposure to different irradiation wavelengths (SI, Figure
SI1). For all compounds the presence of an ESR signal near 335
mT could correspond to some organic radicals, probably radical
cation SP•+. We can assume, according to the literature, that
these compounds were able to change their oxidation state in
the presence of iron cations and upon irradiation.[31–34] Upon
addition of the iron (III) ions to spiropyran, the SP form does not
transform into the MC isomer but converts into radical cation
SP•+ whereas Fe3+ reduces to Fe2+. Upon UV irradiation and in
the presence of the iron (II) ions, SP•+ is reduced to form the
open isomer which forms complexes with Fe3+. It is reported that
the MC– Fe3+ complexes are not reactive or silent to visible light
while the addition of 2,2′-bipyridine, which binds ferric cations
more effectively, leads to usual photochromic reversibility.[31–34]
Synthesis of 1-alkylimidazole derivatives of 5-chloromethylsalicylic
aldehyde 1 - 4: 5-chloromethylsalicylic aldehyde (0.009 mol) has been
dissolved in acetonitrile (10 mL). To the formed solution, 3-alkylimidazole
(0.009 mol) has been added. The mixture has been heated with reflux at
stirring for 6 h. The reaction advance has been checked by TLC. The
product has been filtrated, washed with hexane and ether, and dried. 1
(50.00%), mp: 86 - 90°C, IR (powder): 315, 3091, 2961, 1650, 1592,
1485, 1388, 1346, 1270, 1212, 1147, 1077, 1066, 1057, 973, 963, 948,
937 cm-1.
2 (75.38%), mp: 70 - 73°C, IR (powder): 3142, 3119, 3085, 1846, 1651,
1555, 1484, 1390, 1352, 1324, 1271, 1202, 1147, 1077, 1007, 976, 944,
920 cm-1.
3 (57.76%), mp: 113 - 115°C, IR (powder): 3147, 3094, 2980, 1722, 1651,
1567, 1484, 1390, 1349, 1324, 1275, 1199, 1146, 1087, 1025, 980, 960,
945 cm-1.
4 (64.00%), mp: 134-136 °C, IR (powder): 3148, 3123, 3099, 1721, 1625,
1566, 1485, 1389, 1351, 1323, 1274, 1237, 1201, 1146, 1076, 1066,
1057, 982, 960, 944 cm-1.
We have also investigated the irradiation of compounds in the
presence
of
4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPOL). We could observe a faster decay of TEMPOL in the
presence of spiropyrans with irradiation time (SI, Figure SI2),
comparing with control solution (containing only TEMPOL) that
indicates inhibition of Reactive Oxygen Species such as OH
radicals formed in Fenton reaction. It could be assumed that
nitroxides formation under irradiation could prevent generation of
OH radicals from Fenton reactions by accepting the electron
from the reduced metal complex as shown in the equation
Crystal data for 4: C12H13Cl4FeN2O2, Mr = 414.89, monoclinic, space
group P21/n (no. 14), a = 6.7371(13) Å, b = 8.2079(16) Å, c = 30.517(6) Å,
β = 94.07(3)°, V = 1683.3(6) Å3, Z = 4, T = 298(2) K, Dc = 1.637 g cm−3
,
F(000) = 836, R1 = 0.0404 (191 parameters), wR2 = 0.1015 [I ≥ 2σ(I)],
GOF = 0.914 for all 13017 data. CCDC no. 1859770 (4). The structures
were solved by direct methods and refined by full-matrix least-squares
using the SHELXTL program suite.[36]
below[35,36]
:
Synthesis of spiro compounds 5’-8’: a 1-alkylimidazole derivative of 5-
chloromethylsalicylic aldehyde (1 – 4, 0.0059 mol) has been dissolved in
ethanol 95% (10 mL). 2 drops of catalyst triethylamine have been added,
as well as 1,3,3-trimethyl-2-methyleneindoline (0.0063 mol) freshly
distilled under vacuum. The mixture has been subjected to reflux for 8 h.
The ethanol has been eliminated at the rotary evaporator. The left
precipitate has been washed with hexane and diethyl ether.
H+ +RR NO + Fe2+ RR NOH + Fe3+
Conclusions
The studied organic salts of imidazole derivatives salicylic
aldehyde have appeared to be stronger antiproliferative agents
against cancerous cells than the analogous spiropyrans with
methyl imidazole derivative having higher activity than that of
carboplatinum. At the same time, the toxicity of all studied
compounds has appeared to be lower than that of carboplatin.
Inhibition of ectonucleotidase has been detected as one of the
certain mechanism of antiproliferative activity of the studied
compounds. Moreover, no production of singlet oxygen has
Synthesis of tetrachloroferrate salts of spiro-compounds 5 – 8: one of the
spiro compounds 5’-8’ (0.002 mol) has been dissolved in methanol and
FeCl3·6H2O (0.003 mol) has been added. The mixture has been refluxed
for 5 hours, then the methanol has been eliminated at rotary evaporator
and the left precipitate has been washed with diethyl ether.
6 (81.80%), mp: 165 - 170°C, IR (powder): 2987, 2902, 1590, 1533, 1476,
1451, 1404, 1309, 1260, 1153, 1114, 1076, 1066, 1057, 965, 933 cm-1.
5
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7 (70.71%), mp: 166 - 170°C, IR (powder): 2988, 2902, 1649, 1590, 1532,
1475, 1454, 1405, 1369, 1312, 1261, 1158, 1116, 1076, 1066, 1057, 967,
953, 923 cm-1.
Cell Viability Assays (MTT Assay)
The in-vitro MTT (dimethyl–2–thiazolyl–2,5–diphenyl–2H–tetrazolium
bromide)–based cell viability assay was used for the determination of the
cytotoxic potential of selected derivatives against breast cancer cells i.e.,
MCF-7 and cervical cancer cells i.e., HeLa. The assay was performed
according to a previously described method of Mosmann[41] and Niks and
Otto.[42] Total 90 μL of the cell culture containing 2.5 × 104 cells/ mL were
seeded in 96–well flat–bottom plates. After that, test compound solution
i.e. 10 μL (at a final concentration of 100 µM) was added to the
respective wells and plates were kept for incubation at 37ºC for 24 h. A
100 μL of culture medium without any compound was taken as negative
control. Carboplatin was taken as positive control. After incubation, MTT
reagent (10 μL) was added to each well and again incubated for 4 h at
37ºC. Stopping reagent (100 µL, 50% isopropanol and 50 mL of 10%
sodium dodecyl sulfate) was added to stop the enzyme reaction. The
plates were incubated under agitation at room temperature for an
additional 30 min. Finally change in absorbance was measured at 570
nm after subtracting the background signal (690 nm), using a 96–well
microplate reader (Bio–Tek ELx 800TM, Instruments, Inc. Winooski, VT,
USA). The activity of mitochondrial dehydrogenases of metabolically
active cells resulted in the formation of formazan that represented the
number of viable cells. All experiments were performed in triplicate and
results were reported as percent inhibition values with a mean of three
independent experiments (± SEM).
8 (84.55%), mp: 125 - 130°C, IR (powder): 2973, 2902, 1648, 1589, 1529,
1454, 1404, 1311, 1260, 1156, 1115, 1076, 1066, 1056, 966, 936 cm-1.
Cell Transfection with e5'NT and preparation of membrane fractions
The COS-7 cells were transfected with a plasmid expressing e5'NT
(human and rat)[37] in 10 cm plates by lipofectamine.[38] The confluent
(80-90%) were further incubated for 5 h at 37 °C in simple DMEM
(Dulbecco’s modified Eagle’s medium) containing plasmid DNA (6 µg)
and lipofectamine reagent (24 µL). The reaction was terminated by
diluting the reaction mixture with the same volume of DMEM/F-12
containing foetal bovine serum (20% FBS). Finally the cells were
collected after 40-72 h. The collected cells were washed in triplicate with
cool (4°C) tris-saline buffer and were separated from the harvesting
buffer (0.1 mM phenylmethylsulfonyl fluoride (PMSF), 45 mM Tris buffer
and 95 mM NaCl, pH 7.5) by scraping. This procedure was followed by
the twice centrifugation of these cells at 300×g for 5 min. at 4°C[37]
.
These cells were again suspended in the harvesting buffer comprising
aprotonin (10 µg/mL) and finally sonicated. Cellular and nuclear debris
were removed from the media by centrifugation at 300×g at 4 °C for 10
min. For preservation glycerol (7.5%) was mixed with the resultant
supernatant and stored at -80 °C for further use. Bradford microplate
assay was employed for estimation of protein concentration using bovine
serum albumin as a standard protein.[39]
Cell Cycle Analysis Assay
The cell cycle analysis of the potent anticancer compounds was carried
out as described previously.[43] The cells were incubated with different
compounds for 24 h. Then the cells pellets were formed by centrifugation
at 3000 rpm for 5 min. and cells were again allowed to resuspend in 3%
FBS (200 µL). This step is followed by the addition of propidium iodide (5
µL, 250 µg/mL ), Triton X-100 (5 µL, 10%) and ribonuclease A (25 µL, 10
mg/mL) in each vial and incubated in dark room for 1 h then these
samples were evaluated by BD Accuri flow cytometer and 10,000 events
were collected for each sample. The collected data were analyzed by
using BD Accuri software. Carboplatin was considered as a positive
control and the same procedure was repeated for carboplatin.
Ecto-5'-Nucleotidase Inhibition Assay
The enzyme inhibition assay against human and rat e5'NTs was carried
out according to the previously described method.[40] In this method,
P/ACE MDQ capillary electrophoresis system (Beckman Instruments,
Fullerton, CA, USA) was used that utilized a UV detection system for
evaluation. The selected compounds were prepared in 10% DMSO as a
stock solution with concentration 10 mM and further diluted in assay
buffer (10 mM Tris HCl of pH 7.4 containing 1 mM CaCl2 and 2 mM
MgCl2) to get working solutions (1 mM). The reaction was proceeded
carrying 100 µL of total assay volume with 70 µL of assay buffer, 10 µL of
test compound followed by the addition of 10 µL of h-e5'NT (6.94 ng) or
r-e5'NT (7.17 ng) protein extracts. The mixture was incubated at 37 °C
for 10 min and finally 10 µL of AMP substrate (0.5 mM) was added to
start the reaction. The reaction mixture was allowed to incubate again at
37 °C for 20 min. At the end, the reaction vials were kept in a water bath
(90 °C) for 20 min to denature protein and stop the reaction. Then 50 µL
of each reaction mixture was transferred into a mini CE vial and hydro-
dynamically (0.5 psi) injected into the capillary by taking 5s. The electro-
osmatic separation of peaks i.e., substrate and product happened by
applying a higher voltage (15 kV). The area under its absorbance peak
was calculated for the estimation of product concentration i.e., adenosine
at 260 nm. The compounds that exhibited ≥50 inhibition against either h-
e5'NT or r-e5'NT enzyme were further investigated for their IC50 values.
The experiment was carried out in triplicate by using the above
mentioned experimental procedure.
DNA Interaction Studies
The most potent compounds were further evaluated by performing their
DNA interaction studies by using the previously reported method with
slight modification.[44] The concentrations of DNA were determined at 260
nm by using micro-plate reader FLUOstar Omega. By using the molar
absorption coefficient of 6600 M-1cm-1, the concentration of DNA was
found as 4.15×10-4 M at 260 nm. Then different concentrations of DNA
i.e. 0, 40, 90, 140, 190, 240, 290, and 340 µM were allowed to interact
with fixed concentrations of compound 4 i.e., 40 µM and their reference
solution. Then plates were allowed to incubate for 30 min. at room
temperature. The absorption spectra were measured by using 96-well
plates of 5.5 mm path length with blank correction. All the experiments
were performed in triplicate. The mode of interaction of compounds was
determined by using Omega–Data Analysis Software, Program Version:
3.00 R3. DNA interaction studies for carboplatin as a positive control was
also performed.
Cell lines and cell cultures
Evaluation of Apoptosis by fluorescence microscopy
HeLa cells, MCF-7 cells and BHK-21 cells were cultured in DMEM
medium supplemented with penicillin (100 U/mL), streptomycin (100
μg/mL) and heat-inactivated FBS (10%). Similarly k-562 cells were
cultured in RPMI-1640 medium containing the same additional
composition as earlier. These cell lines were cultured and incubated in
separate culture flasks at 37 ºC in 5% CO2 incubator. The confluent
monolayer cells (1×104 cells/mL) were seeded in 96-well plate to conduct
initial cytotoxicity experiments.
Morphological analysis of apoptosis was determined by using 4',6-
diamidino-2-phenylindole (DAPI) and propidium iodide (PI) dyes. The
specified amounts of cells (1.2×106) were cultured in 24-well plates along
with coverslips overnight. Next day, 100 µM of the most potent
compound i.e. 4 (IC50 =1.14 µM) was treated with the cells along with the
negative and positive control i.e. carboplatin. The plate was allowed to
incubate at 37ºC in CO2 (5%) for 24 h. After incubation the cells were
fixed and washed with Formalin (4%) and PBS (pH 7), respectively.
6
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10.1002/cmdc.201800520
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FULL PAPER
Finally, the cells were stained with DAPI or PI (10µg/ml) and incubated
for 10 min in dark at room temperature and then examined under a
fluorescence microscope (Nikon Eclipse-Ni Japan) at an excitation (358
nm) and emission (461 nm) wavelengths.[45]
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Acknowledgements
V.B. and F.M. are grateful for the funding support offered by the Science
and Technology Center in Ukraine and the National Agency for Research
and Development of the Republic of Moldova under international project
17.80013.8007.10/6245STCU. F.M. and D.P. are grateful for the funding
offered by the BMBF project 01DK13029 and Prof. Dr. Annie K. Powell
(Karlsruhe Institute of Technology, Germany) for generous support. V.B.
and D.P. are grateful for the generous support from the Alexander von
Humboldt Foundation. J.I. is grateful to the Higher Education
Commission of Pakistan for the financial support through Project No. 20-
3733/NRPU/R&D/ 14/520. The authors acknowledge the contributions
and are grateful to Dr. A. Paul (ESR facilities) from the BAM Federal
Institute for Materials Research and Testing, Berlin, Germany. The
authors also gratefully acknowledge Prof. Dr. George E. Kostakis
(University of Sussex, UK) for help with preliminary single crystal x-ray
diffraction experiments.
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Keywords: cancer
•
ionic liquids
•
organic salts
•
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Entry for the Table of Contents
The reported tetrachloroferrate salt of 1-methylimidazole derivative of salicylic aldehyde has higher cytotoxicity towards HeLa cells
and lower cytotoxicity towards BHK-21 cells as compared to carboplatinum. The compound induces stronger inhibition of
ectonucleotidase than carboplatinum, the effect being one of the main mechanisms of its antiproliferative activity.
9
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