Synthesis and evaluation of pyridinium-hydrazone derivatives as potential antitumoral agents

Article abstract of DOI:10.1111/cbdd.13177

The hydrazones of 4-hydrazinylpyridinium bearing alkylphenyl groups on pyridinium nitrogen were synthesized and evaluated for their cytotoxic activity against MCF-7, PC3, U2OS, and HEK293 cell lines by Wst1 cell proliferation assay. Cytotoxic activity results indicated that d derivatives having butylene chain; 4 and 5 series having naphthalene and anthracene ring systems showed high cytotoxic activity (IC₅₀?=?3.27–8.54?μm) on cancer cells. 3d (4-(2-(4-hydroxybenzylidene)hydrazinyl)-1-(4-phenylbutyl)pyridinium bromide) was the most cytotoxic compound with IC₅₀ value of 3.27?μm against MCF-7. The most active derivatives (1d, 2d, 3d, 4, and 5 series) were selected to investigate for the effects on autophagy by analyzing the expression of autophagy marker proteins. The conversion of LC3-I to its lipidated form LC3-II is essential for autophagy and related to autophagosomes. According to our results, all tested compounds except for 3d induced lipidated form LC3-II accumulation. Then, the effects of the compounds on p62 protein level were also analyzed by the immunoblotting as the autophagy inhibition results in accumulation of p62. Further molecular mechanistic studies including morphological analysis and live–death assays indicated that all tested compounds (1d, 2d, 3d, 4, and 5 series) are potent antitumoral molecules and all except for 3d have potential to inhibit autophagic flux.

Full text of DOI:10.1111/cbdd.13177

DR VILDAN ALPTUZUN (Orcid ID : 0000-0002-1477-4440)
Article type : Research Article
Synthesis and Evaluation of Pyridinium-Hydrazone Derivatives
as Potential Antitumoral Agents
Sülünay Parlar1, Yalçın Erzurumlu2, Recep Ilhan2, Petek Ballar Kırmızıbayrak2,
Vildan Alptüzün1*, Ercin Erciyas1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, Bornova,
Izmir, Turkey
2Department of Biochemistry, Faculty of Pharmacy, Ege University, Bornova, Izmir, Turkey
*Correspondence
Vildan ALPTÜZÜN
Department of Pharmaceutical Chemistry
Faculty of Pharmacy, Ege University
35040 Bornova, Izmir-Turkey
Tel: +90 232 311 40 77
Fax: +90 232 388 52 58
Email: vildan.alptuzun@ege.edu.tr
ABSTRACT
The hydrazones of 4-hydrazinylpyridinium bearing alkylphenyl groups on pyridinium
nitrogen were synthesized and evaluated for their cytotoxic activity against MCF-7, PC3,
U2OS and HEK293 cell lines by Wst1 cell proliferation assay.
Cytotoxic activity results indicated that d derivatives having butylene chain; 4 and 5 series
having naphthalene and anthracene ring systems, showed high cytotoxic activity (IC50= 3.27-
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13177
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8.54 M) on cancer cells. 3d (4-(2-(4-hydroxybenzylidene)hydrazinyl)-1-(4-
phenylbutyl)pyridinium bromide) was the most cytotoxic compound with IC50 value of 3.27
μM against MCF-7. The most active derivatives (1d, 2d, 3d, 4 and 5 series) were selected to
investigate for the effects on autophagy by analyzing the expression of autophagy marker
proteins. The conversion of LC3-I to its lipidated form LC3-II is essential for autophagy and
related to autophagosomes. According to our results, all tested compounds except for 3d,
induced lipidated form LC3-II accumulation. Then, the effects of the compounds on p62
protein level were also analyzed by the immunoblotting since the autophagy inhibition results
in accumulation of p62.
Further molecular mechanistic studies including morphological analysis and live death assays
indicated that all tested compounds (1d, 2d, 3d, 4 and 5 series) are potent antitumoral
molecules and all except for 3d have potential to inhibit autophagic flux.
KEYWORDS
pyridinium salts, hydrazone, anticancer activity, autophagy, p62
1 INTRODUCTION
Cancer is one of the causes of death affecting a significant amount of the population
worldwide. The number of patients dealing with cancer increases every year. According to
the International Agency for Research on Cancer reports, 14.1 million new cancer cases, 32.6
million people suffering from cancer (within 5 years of diagnosis) and 8.2 million cancer
deaths were recorded in 2012 worldwide and it is estimated that deaths resulting from cancer
will reach to 21.1 million by 2030.[1]
Although several chemotherapeutic agents have been used for the cancer treatment;
serious side effects, high toxicity and the drug resistance limit their use in the treatment.
Depending on elucidation of molecular mechanisms concerning cancer, new strategies have
been developed for the treatment of this disease. Recently, researchers focus on discovering
effective molecules targeting cell death pathways such as apoptosis, necrosis and
autophagy.[2]
Apoptosis and autophagy are the main types of programmed cell death (PCD) related to
cancer and its treatment.[3] Apoptosis is the best-characterized type of PCD and it has been
targeted to prevent and treat cancer for decades. Exploring other types of PCDs creates new
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opportunities for cancer treatment; especially when targeting apoptosis is not effective for
cancer treatment in conditions seen in apoptosis-resistant cancer cells.[4] Autophagy is a
physiological process that maintain cellular homeostasis by degrading and recycling
organelles and proteins.[5] Autophagic defect may constitute metabolic stress, genetic damage
susceptibility and later cancer promoting environment in cells. On the contrary, autophagy
may increase stress tolerance and survival in cancer cells under adverse conditions. Thus,
autophagy has a context-dependent role in cancer, and both induction and inhibition of
autophagy may be the target of cancer therapy.[6, 7]
Hydrazones are a special group of the Schiff bases. Hydrazone derivatives have
important biological activities and this functional group is found as a core structure in many
chemotherapeutic agents having antimicrobial, antimalarial, antileishmanial, antiviral and
anticancer effects.[8-10] Many researchers have reported hydrazones as promising anticancer
agents.[11-18] According to the reports, these hydrazone compounds display anticancer activity
via diverse molecular mechanisms.[l6-21] Among them, thiazole-hydrazone derivative
(CPTH6) was found to have anticancer potency via apoptosis and autophagy.[22]
Researchers have also carried out studies for novel anticancer agents with quaternary
nitrogen. These studies have revealed that the cationic charge might play a role not only for
interacting with the target, but may also enhance water solubility, cell membrane
permeability and cellular uptake.[23-26]
Many studies have shown that some of the antimicrobial compounds exhibit cytotoxic
activity against cancer cells.[27-29] In our previous studies, oxime-ether and hydrazone
derivatives with quaternary pyridinium core were found to have remarkable antimicrobial and
antileishmanial activities.[30-32]
On the other hand, 1,4-dihydropyridine derivatives obtained from a new series
pyridinium-hydrazones by using appropriate base[33] were evaluated as AChE inhibitory
activity by our group.[34] In the light of the facts mentioned above, our main goal was to
investigate anticancer profile of these pyridinium-hydrazone compounds with this study. We
synthesized the pyridinium-hydrazone derivatives having an aromatic moiety connected to
the pyridinium nitrogen via alkyl linkers of different lenght. At the opposite end of the
molecule nonsubstituted-, p-hydroxy-, and p-nitrobenzaldehydes were selected as aldehyde
function to form their hydrazones. Also, to investigate the effects of planar aromatic groups
on the activity, 1-naphthaldehyde and 9-anthraldehyde derivatives were added to the series.
Then, we planned to screen the cytotoxic activity on different cell lines.
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Numerous studies identified that pyridinium or hydrazone containing molecules
exhibited effects on autophagy. Compound VLX600 bearing pyridine-2-hydrazone moiety[35]
and compound CCCP (carbonyl cyanide m-chlorophenylhydrazone)[36] were reported to have
the effect on autophagy process. Besides, compounds LCL124 and C18-pyridinium
ceramides having a long hydrophobic alkyl chain on quaternary pyridinium nitrogen were
found to play a role on autophagy.[37, 38] On the other hand, chloroquine (CQ), lucanthone and
acridine derivatives including planar ring system are the autophagic inhibitors under
investigation as anticancer agents.[39-42] Due to these core structures’ role on autophagy, as a
further mechanistic study, we designed to test the effects on autophagy and ubiquitin
proteasome system of the most active compounds.
2 METHODS AND MATERIALS
2.1 Chemistry
All melting points were determined using an Electrothermal IA9100 melting point apparatus
(Electrothermal, Essex, U.K.) and are not corrected. The infrared (IR) spectra of the
compounds were monitored by attenuated total reflectance (ATR) (PerkinElmer Spectrum
100 FT-IR, Waltham, MA, USA). 1H- and 13C-NMR spectra were recorded with a Varian AS
400 Mercury Plus NMR spectrometer (Varian, Palo Alto, CA, U.S.A.) operated at 400 and
1
100 MHz for H and 13C, respectively. Tetramethylsilane (TMS) was used as an internal
standard and DMSO-d6 was used as a solvent. Chemical shifts were measured in parts per
million (δ). The J values were given in Hz. Abbreviations for data quoted are: s, singlet; d,
doublet; t, triplet; quin, quintet; dd, doublet of doublets; m, multiplet; bs, broad singlet. All
chemicals, reagents and solvents used for synthesis were high-grade commercial products and
they were purchased from Sigma, Acros and Merck companies. LC/MS was recorded on a
Thermo MSQ Plus (San Jose, CA, USA) mass spectrometer using ESI. Elemental analyses
(C, H, N) were performed by Leco TruSpec Micro (Leco, St. Joseph, MI, USA). Analytical
thin layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel 60 F254) with
detection by UV light (254 nm).
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2.2 General procedure for the synthesis of compounds 1a-5d
The title compounds, except for 3 series, were previously synthesized by our group.[34] They
were prepared as shown in Scheme 1. In the first step, 4-chloropyridine (0.1 mol) and
hydrazine monohydrate (0.2 mol) in 1-propanol (30 ml) was refluxed for 18 h. In the second
step, 4-hydrazinylpyridine (0.033 mol) was condensed with 0.04 mol various aromatic
aldehydes (benzaldehyde, p-nitrobenzaldehyde, p-hydroxybenzaldehyde, 1-naphthaldehyde,
9-anthraldehyde) for 6-18 h in 1-propanol to yield the corresponding hydrazone derivatives
(1-5). Finally, the title compounds (1a-5d) were obtained by quaternization of the pyridine-
hydrazone derivatives (0.01 mol) with 0.02 mol appropriate substituted alkyl halide (benzyl
chloride, 2-phenethyl bromide, 3-phenylpropyl bromide, 4-phenylbutyl bromide) in ethanol
under reflux for 1-16 h. The precipitate was filtered and washed with cool ethanol and
crystallized from ethanol to give the final compounds (1a-5d). The spectral data of all
compounds were reported for the first time with this study (supplementary data). Compounds
3a-3d are original.
2.3 Biological activities
2.3.1 Cell culture
Three cancer cell lines namely PC3 (human prostate cancer), MCF-7 (human breast cancer),
U2OS (human osteosarcoma) and one non-tumoral cell line HEK293 cells (human embryonic
kidney cells) were included in the study. Cell lines were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and maintained in a
humidifier incubator at 37 °C supplied with 5% CO2. All the reagents were purchased from
Biological Industries (Israel).
2.3.2 Wst1 cell proliferation assay
PC3, MCF-7, U2OS and HEK293 cells were seeded in 96-well plates (5000 cells/well). All
tested compounds were dissolved in DMSO. 24 hours later, cells were treated with the
compounds from the library with the final concentrations of 1, 2, 5, 10, 25 μM. DMSO and
doxorubicin were used as negative solvent control and positive cytotoxic control,
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respectively. In all samples DMSO concentration was 0.2%. Following 48 hours incubation,
cell viability was determined by the colorimetric WST-1 assay (Roche) as indicated in the
protocol provided by manufacturer. Formazan dye formation was evaluated using a scanning
multiplate spectrophotometer (Varioscan, Thermo) at 450 nm and a 620-nm reference filter.
To determine the IC50 values, a sigmoid-dose response curve was fitted to the data using
nonlinear regression in GraphPad Prism 5 software.
2.3.3 Immunoblotting
U2OS cells were treated with the IC50 concentrations of compounds or DMSO control for 24
h. Equal amounts of cell lysates were loaded to the SDS-PAGE gels. Following
electrophoresis, proteins were transferred to the PVDF membrane (Millipore). After blocking
with 5% nonfat milk in PBS-T buffer, membranes were blotted with primary antibody for 1 h
at room temperature. After 3 times of washing procedure membranes were incubated with
secondary antibody for 1 h at room temperature. Visualization of the results were done using
Clarity ECL Solution (BioRad) and Fusion FX (Vilber Lorurmat). Actin was used as loading
control. Anti-LC3A/B, anti-p62 and anti-K48 specific polyubiquitin antibodies were
purchased from Cell Signaling, while anti-actin antibody was from Sigma.
2.3.4 DAPI staining
U2OS cells were inoculated on coverslips in 12-well plates for 24 h. Then the cells were
treated with IC50 concentrations of the compounds for 24 h. To determine nuclear
condensation by DAPI (Sigma Aldrich) staining, cells were fixed with 4% paraformaldehyde
for 30 min at 4 °C and washed six times with ice cold PBS. Then coverslips were incubated
with 0.5 μg/ml DAPI (4',6-diamidino-2-phenylindole) for 5 min in dark and examined under
fluorescent microscope (Olympus IX71 fluorescent microscope).
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2.3.5 Live Dead Cell staining
In order to distinguish between health live and dying apoptotic/necrotic cells Live-Dead Cell
Staining Kit (Biovision) was utilized considering the protocol of manufacturer. U2OS cells
were inoculated on coverslips in 12-well plates for 24 h. Then the cells were treated with IC50
concentrations of the compounds for 24 h. This kit includes Live-Dye™, a cell-permeable
green fluorescent dye, to stain live cells and propidium iodide (PI), a cell non-permeable red
fluorescent dye to stain dying cells. Following incubation with the Staining Solution,
coverslip was inverted on a glass slide and cells were visualized by fluorescence microscopy
(Olympus IX71 fluorescent microscope).
3 RESULTS AND DISCUSSION
3.1 Chemistry
In this study, twenty 4-(2-arylidinehydrazinyl)pyridinium derivatives were synthesized
according to mainly reported method.[33] The synthetic route is given in Scheme 1.
Firstly, 4-hydrazinylpyridine was furnished by nucleophilic substitution of hydrazine
with 4-chloropyridine. Secondly, 4-hydrazinylpyridine and corresponding aromatic aldehydes
were reacted in 1-propanol to give hydrazone derivatives (1-5). Finally, the title compounds
1a-5d were achieved by quaternization of intermediate compounds (1-5) with alkyl halides in
ethanol under reflux condition. These final compounds are very sensitive to convert into the
1,4-dihyropyridines in the presence of appropriate base such as NaOH with a good yield.[34]
1
The structures of the compounds were confirmed by spectral (IR, H NMR, 13C NMR
and ESI Mass) and elemental analyses. The purity levels of compounds were determined by
1
elemental analyses (C, H, N) and the results were within ±0.4% of the calculated values. H
NMR spectra of the title compounds were consistent with expected resonance signals in
terms of chemical shifts and integrations. All the 13C NMR findings confirmed the structures
proposed. The mass spectra of the title compounds were recorded in respect to the positive
ion mode electrospray ionization (ESI+) technique. The M+ ions of title compounds
compromised with the calculated molecular weights.
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3.2 Biological evaluation
The cytotoxic activity of the title compounds was evaluated against human cancer cell lines
including MCF-7, PC3, U2OS and one non-tumoral cell line, HEK293, by using Wst1 cell
proliferation assay. Doxorubicin was used as the reference compound. IC50 values of the title
compounds were listed in Table 1.
In our previous study, the antimicrobial activity results of the pyridinium-hydrazone
derivatives indicated that the longer the side chain on pyridinium nitrogen, the more
antimicrobial activity it possesses.[30] Since various studies have revealed that some of the
antimicrobial compounds having toxicity on bacteria display cytotoxic activity against cancer
cells[27-29], we evaluated the effect of the distance between the pyridinium nitrogen and the
phenyl ring on the cytotoxic activity. According to the activity results, among the
nonsubstituted and the substituted phenyl series (1a-1d, 2a-2d, 3a-3d); generally, d
derivatives (1d, 2d, 3d) having butylene chain displayed the highest cytotoxic activity
whereas a derivatives (1a, 2a, 3a) having methylene chain exhibited poor cytotoxicity against
the studied cell lines. Moreover, d derivatives have approximately two times more
cytotoxicity than the c derivatives on especially U2OS and MCF-7 cell lines. Unlike phenyl
series, all naphthalene (4) and anthracene (5) series (4a-4d, 5a-5d) exhibited high cytotoxic
activity.
Many studies indicated that planar compounds exhibited significant cytotoxic activity.[43-
45]
Then, we evaluated the effect of planar ring on cytotoxic activity. According to the
cytotoxic activity results, it is obvious that replacement of the phenyl moiety with
naphthalene (4) and anthracene (5) ring systems remarkably improved the cytotoxic activity
for the derivatives especially bearing benzyl side chain. All compounds (IC50= 3.33-7.54 µM)
of series 4 and 5 displayed comparable cytotoxic activity on cancer cells to the reference
compound, doxorubicin (IC50= 1.89-2.88 µM).
The cytotoxic activity results suggest that the distance between the pyridinium nitrogen
and the phenyl ring is critical for the activity in the series 1, 2, 3, and generally the planarity
of the ring system connecting to the hydrazone moiety positively influences the cytotoxic
activity. On the other hand, the substitutions at para position of the phenyl ring connected to
the hydrazone moiety did not lead to significant change in the cytotoxic activity. Among the
tested compounds, series 3 bearing hydroxyl substituent displayed higher cytotoxic activity
on MCF-7 cell line and nitrosubstituted phenyl derivatives (series 2) showed better selectivity
on MCF-7, PC3, U2OS cell lines compared to non-tumoral HEK293 cells. Besides, d
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derivatives exhibited higher cytotoxic activity against U2OS than the other cell lines. Among
the tested compounds, derivatives with the highest cytotoxic activity (1d, 2d, 3d, 4 and 5
series) were selected to further investigate their mechanism of activity.
Autophagy, a catabolic pathway that eliminates long-lived proteins and organelles, is one
of the important targeted mechanisms in the treatment of cancer disease. While autophagy
maintains at basal level under normal nutrient conditions, it can be upregulated to continue
cell survival under unexpected conditions such as starvation, hypoxic stress. Autophagy
serves as tumor suppressive or tumor inducer depending on the tumor stage. Autophagy can
suppress the development of cancer in the early stage, whereas it can contributes to cancer
cell survival in the formed cancer.[46, 47]
Several proteins are required for the formation of the autophagosome. One of these
proteins is the microtubule-associated light chain 3 (LC3). During the autophagy, LC3 is
hydrolyzed to LC3-I and then the conjugation of LC3-I with phosphatidylethanolamine
produce the lipidated form LC3-II.[48, 49] Thus, LC3 is widely used marker of autophagy. We
firstly evaluated the autophagy activity by the conversion of LC3-I to LC3-II by immunoblot
analysis in U2OS cell. DMSO was used as a positive control. Bafilomycin A1 was used as
the standard autophagy inhibitor compound. The amount of LC3-II correlates with the
number of autophagosomes. Immunoblot analysis showed that LC3-II was accumulated in
these compounds except for 3d (Figure 1A). Besides LC3 conversion, accumulation of p62 is
also commonly used as a autophagy marker. p62 is a substrate of the autophagic process and
degraded by autophagy. Accumulation of p62 is related to the inhibition of autophagy flux.[50,
51]
Thus, we also determined the amount of p62 to investigate the role of the selected
compounds in autophagy (Figure 1A). The immunoblot analysis data revealed that p62 levels
increased as a result of treatment the cells with the compounds, again except for 3d (Figure
1A and 1B). These results indicate that the compounds are presumably potent autophagic flux
inhibitors. They may be effective in the formed cancer due to their inhibitory effect on
autophagy.
It is known that there are two major protein degradation pathway in cell, namely
autophagy and ubiquitin proteasome system. Since we found some significant effect on
autophagy, we also tested the effect of compounds on the ubiquitin mediated degradation
monitoring total K48-linked polyubiquitinated proteins. MG132 was used as the standard
proteasome inhibitor. Our data suggests that only some of our compounds slightly caused
accumulation of total K48-linked polyubiquitinated proteins (Figure 1C and 1D).
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Next, we prompted to investigate the effect of selected compounds (1d, 2d, 3d, 4d and
5d) on the cell and nuclear morphology, using light and fluorescence microscopy,
respectively. As seen in Figure 2A, 1d, 2d, 4d and 5d severely changed the morphology of
cells. It is also noteworthy to mention that these compounds, except for 3d, caused formation
of intracytoplasmic vacuoles. It is known that cells undergoing apoptosis demonstrates
nuclear condensation and DNA fragmentation. Thus, we also analyzed the effect of tested
compounds for their nuclear staining using DAPI and fluorescent microscopy. While control
cells show regular nuclear DAPI staining with uniform distribution, cells treated with tested
compounds showed fragmented nuclei (Figure 2B). Next, we wanted to distinguish between
live cells and apoptotic/necrotic cells on compounds treated U2OS cells and our data
indicates that while there is no PI staining observed in DMSO treated cells, 1d, 2d, 3d, 4d
and 5d treated cells all have significant PI staining (Figure 2C).
CONCLUSIONS
In this study, we assessed the anticancer potencies of 20 pyridinium-hydrazone derivatives
against three different types of cancer cell lines. Almost all compounds were able to display
cytotoxic activity in micromolar range of concentration. According to cytotoxic activity
results, it can be concluded that the distance between the pyridinium nitrogen and the phenyl
ring is critical for the activity in the series 1, 2, 3, and generally, the planarity of the ring
system connecting to the hydrazone moiety positively influences the cytotoxic activity. In
addition, further mechanistic study revealed that all the tested compounds except for 3d
exhibited inhibitory effect on autophagy and some of them slightly showed proteasome
inhibitory activity. It can be suggested that compounds 1d, 2d, 4 series (4a, 4b, 4c, 4d) and 5
series (5a, 5b, 5c, 5d) are presumably potentially autophagic flux inhibitors.
ACKNOWLEDGEMENTS
This study was supported by Ege University Scientific Research Project Council (Project
numbers: 10/ECZ/012 and 10/ECZ/028). The authors would like to thank Professor Dr.
Ulrike Holzgrabe (University of Würzburg) for her valuable discussion.
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CONFLICT OF INTEREST
The authors report no conflict of interests.
SUPPORTING INFORMATION
Supporting Information can be found in the online version of this article:
Spectral data of final compounds
1H NMR spectra of final compounds
13C NMR spectra of final compounds
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A LIST OF CAPTIONS
SCHEME 1 Synthesis of the title compounds
TABLE 1 Cytotoxic activities of the title compounds
FIGURE 1 A. U2OS cells were treated with compounds at IC50 doses for 24 h. At the end
treatment cells were lysed and the autophagic marker proteins LC3 and p62 were analyzed by
immunoblotting. The conversion of LC3-I and -II is shown. Actin was used as loading
control. Bafilomycin (Baf) was used as standard autophagy inhibitor control. B. The
accumulation of p62 was analyzed using the densitometric analyses of three independent
assays using ImageJ software (http://imagej.nih.gov/ij/). The fold increase compared to
DMSO treated control cell group was presented in the graph. C. U2OS cells were treated
with compounds at IC50 doses. 24 hours later cells were harvested and analyzed via SDS-
PAGE followed by immunoblotting. The total K48-linked ubiquitined protein levels were
determined using a specific antibody against K48-linked ubiquitin chains. Mg132 was used
as standard proteasome inhibitor. D. Densitometric analyses of K48-linked polyubiquitinated
proteins were performed using ImageJ software (http://imagej.nih.gov/ij/). The data from
three independent experiments was analyzed and the fold increase compared to DMSO
treated control cell group was presented in the graph.
FIGURE 2 A. Live images of U2OS cells treated with compounds indicated compounds and
DMSO control for 24 h and visualized under inverted microscope. B. DAPI staining of cells
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treated with indicated compounds at IC50 concentrations for 24 h. Arrows indicates nuclear
fragmentation. C. Live/dead cell staining of cells treated with indicated compounds at IC50
concentrations for 24 h. Green: Live-Dye™, Red: propidium iodide (PI).
SCHEME 1 Synthesis of the title compounds
This article is protected by copyright. All rights reserved.

TABLE 1 Cytotoxic activities of the title compounds
IC50 ± SD (µM)a
Compound
R
n
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
-
U2OS
MCF-7
PC3
HEK293
25>
phenyl
25>
25>
25>
25>
25>
1a
phenyl
25>
20.97 ± 1.41
10.79 ± 0.40
1b
phenyl
10.21 ± 0.09 9.07 ± 0.04
4.71 ± 0.05 5.75 ± 0.24
19.33 ± 3.89 19.46 ± 1.05 18.60 ± 0.64 25>
7.8 ± 0.10
1c
phenyl
8.54 ± 0.30 7.98 ± 0.31
1d
p-nitrophenyl
p-nitrophenyl
p-nitrophenyl
p-nitrophenyl
p-hydroxyphenyl
p-hydroxyphenyl
p-hydroxyphenyl
p-hydroxyphenyl
naphthalene-1-yl
naphthalene-1-yl
naphthalene-1-yl
naphthalene-1-yl
anthracene-9-yl
anthracene-9-yl
anthracene-9-yl
anthracene-9-yl
-
2a
10.26 ± 0.19 8.39 ± 0.50
8.87 ± 0.07 22.50 ± 1.01
2b
12.67 ± 0.55 11.67 ± 0.47 8.84 ± 0.15 18.26 ± 0.90
2c
5.21 ± 0.25
25>
5.59 ± 0.37
6.71 ± 0.16 9.60 ± 0.19
2d
22.18 ± 2.98 25>
25>
3a
19.53 ± 1.24 4.67 ± 0.34
10.41 ± 0.57 6.40 ± 0.52
13.11 ± 2.31 25>
3b
9.95 ± 0.19 10.82 ± 0.08
7.86 ± 0.37 8.05 ± 0.77
5.32 ± 0.50 8.21 ± 0.04
6.03 ± 0.25 7.72 ± 0.47
6.16 ± 0.29 7.50 ± 0.40
6.10 ± 0.23 8.92 ± 0.20
3.61 ± 0.04 6.28 ± 0.24
3.99 ± 0.10 6.99 ± 0.14
4.09 ± 0.04 5.31 ± 0.18
3.91 ± 0.11 4.57 ± 0.14
2.88 ± 0.19 1.10 ± 0.07
3c
3.47 ± 0.73
5.53 ± 0.23
6.92 ± 0.30
6.39 ± 0.34
4.64 ± 0.05
3.73 ± 0.13
4.41 ± 0.06
3.65 ± 0.10
3.33 ± 0.10
1.89 ± 0.28
3.27 ± 0.38
7.54 ± 0.33
5.83 ± 0.45
6.11 ± 0.26
6.44 ± 0.66
4.93 ± 0.06
6.84 ± 0.42
5.04 ± 0.12
3.57 ± 0.33
1.99 ± 0.10
3d
4a
4b
4c
4d
5a
5b
5c
5d
Doxorubicin
aValues represent mean ± SD of three independent experiments.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.