Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

New hydrazide-hydrazones and 1,3-thiazolidin-4-ones with 3-hydroxy-2- T

naphthoic moiety: Synthesis, in vitro and in vivo studies
⁎
Łukasz Popiołeka, , Iwona Piątkowska-Chmielb, Monika Gawrońska-Grzywaczb,
Anna Biernasiukc, Magdalena Izdebskab, Mariola Herbetb, Marcin Sysab, Anna Malmc,
Jarosław Dudkab, Monika Wujeca
a
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, 4A Chodźki Street, 20-093 Lublin, Poland
b
Department of Toxicology, Faculty of Pharmacy, Medical University of Lublin, 8B Jaczewskiego Street, 20-090 Lublin, Poland
c
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, 1 Chodźki Street, 20-093 Lublin, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: In this research we synthesized, identified and evaluated new hydrazide-hydrazones (1–3) and 1,3-thiazolidin-4-
Hydrazide-hydrazone one derivatives (4–6) for in vitro and in vivo activity. New hydrazide-hydrazones (1–3) were obtained by the
1,3-Thiazolidin-4-one condensation reaction of 3-hydroxy-2-naphthoic acid hydrazide with appropriate aldehydes. Synthesized hy-
Antiproliferative activity drazide-hydrazones (1–3) were subjected to cyclization reaction with mercaptoacetic acid which afforded with
Analgesic activity
new 1,3-thiazolidin-4-one derivatives (4–6). Among 1,3-thiazolidin-4-one derivatives tested (4–6), compound 6
Antimicrobial activity
exhibited highest and most selective cytotoxicity towards human renal adenocarcinoma cells (769-P) and it did
not affect the growth of normal cells (H9c2, GMK). Whereas its hydrazide-hydrazone (compound 3) showed
significant antiproliferative activity against both tested human cancer cell lines: renal adenocarcinoma (769-P)
and hepatocellular carcinoma (HepG2), however with less selectivity. The in vivo studies focused on the anti-
nociceptive activity of newly synthesized 1,3-thiazolidin-4-one derivatives (4–6). The preliminary screening of
novel compounds showed that 1,3-thiazolidin-4-one derivatives (4–6) are safe and not toxic against CNS of mice.
Among tested derivatives one compound (6) displayed significant analgesic activity.

1. Introduction
The hydrazide-hydrazones and 1,3-thiazolidin-4-one derivatives
constitute important classes of organic compounds which attracted
significant continuing interest over the years due to their promising
biological activities [1–3].
The hydrazide-hydrazones, despite being biologically active, mainly
as chemotherapeutic agents, are also found to be very useful as inter-
mediates for the synthesis of a variety of the heterocyclic compounds,
e.g. 1,3-thiazolidin-4-ones [1].
The 1,3-thiazolidin-4-one derivatives are a very intriguing chemical
group, as they serve as a core of many well-known pharmaceuticals and
possess wide spectrum of biological activity such as, antitubercular
[2,3], antibacterial [2,4–6], antiviral [2,6–8], anti-inflammatory [6,9]
and analgesic properties [6].
Despite high availability of painkillers, the pain control is still a
therapeutic challenge in the world, mainly because of its hetero-
geneity. The pain control system based on polytherapy increases the
unpleasant side effects of some medications. Additionally, in some
cases even the strongest painkillers in dangerously high doses are
unable to cope with the pain. Taking into account the above facts we
undertook the synthesis of compounds with already proven biolo-
gical activity [6]. The 1,3-thiazolidin-4-ones and hydrazide-hy-
drazones are still attracting attention due to their antiproliferative
and antitumor activities [1,2,6]. Cancer as a multifactorial disease
represents one of the most important cause of mortality in developed
countries. Kidney as well as liver cancers are among the top ten most
common malignant tumors in the world. In both types of cancers, the
morbidity rates tend to increase and the cancers have very high
fatality rates. In addition, the mortality rates of hepatocellular car-
cinoma (HCC) are even higher than the incidence rates because the
liver is a preferred site of metastasis for many cancers. Therefore,
HCC is the third leading cause of death globally [10,11].
In view of aforementioned facts and in continuation of our research
interests concerning the biologically active organic compounds, the aim
of this study was to design and synthesize hydrazide-hydrazones and
2,3-disubstituted 1,3-thiazolidin-4-one derivatives and subsequently to
test their possible activities in in vitro and in vivo experiments. The

⁎
Corresponding author.
E-mail address: lukasz.popiolek@umlub.pl (Ł. Popiołek).

https://doi.org/10.1016/j.biopha.2018.04.163
Received 20 October 2017; Received in revised form 20 April 2018; Accepted 23 April 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Ł. Popiołek et al.
multi-target compounds are very relevant in drug discovery process and
of interest to current medicinal chemistry.
2. Materials and methods
2.1. Chemistry
Reagents and solvents used in this research were obtained from
Sigma-Aldrich (Munich, Germany) and Merck Co. (Darmstadt,
Germany). Melting points were determined on Fisher-Johns blocks
melting point apparatus (Fisher Scientific, Germany) and presented
without correction. The UV–VIS spectra were performed with the use of
HITACHI U-2001 spectrophotometer and DMSO as a solvent. The 1H
NMR and 13C NMR spectra have been performed with the use of Bruker
Avance 300 apparatus (Bruker BioSpin GmbH, Germany) in DMSO-d6
and with TMS used as the internal standard. Chemical shifts are pre-
sented in ppm (δ) and the coupling constants (J) are given in Hertz.
Thin layer chromatography (TLC) was performed on pre-coated alu-
minum sheet 60 F254 plates (Merck Co. USA) and used to monitor the
progress of the reactions and the purity of obtained compounds (solvent
system: CHCl3/C2H5OH 10:1, v/v). The spots were detected by ex-
posure to the UV lamp at 254 nm. The elemental analysis of synthesized
compounds was carried out by AMZ 851 CHX analyzer (PG, Gdańsk,
Poland). The results of elemental analysis (C, H, N) were within ±
0.4% of the calculated values.
2.1.1. Synthesis of hydrazide-hydrazones of 3-hydroxy-2-naphthoic acid
(1–3)
Hydrazide-hydrazone derivatives (1–3) were obtained on the basis
of the procedure described earlier [12]. 0.01 mol of 3-hydroxy-2-
naphthoic acid hydrazide (H) was dissolved in 10 mL of ethanol (96%)
and 0.011 mol of appropriate substituted aromatic aldehyde was added.
The solution was heated under reflux for 3 h. After that, the solution
was allowed to cool and then put to the refrigerator for 24 h. Subse-
quently, the formed precipitate was filtered off and re-crystallized from
ethanol.
N-[(2-chlorophenyl)methylidene]-3-hydroxynaphthalene-2-carbo-
hydrazide (1)
Yield: 96%; m.p.: 247–249 °C. 1H NMR (DMSO- d6) δ
(ppm) = 7.32–7.39 (m, 2 H, ArH), 7.47–7.58 (m, 5 H, ArH), 7.76–7.79
(d, 1 H, ArH, J = 9 Hz), 7.91–7.93 (d, 1 H, ArH, J = 6 Hz), 8.05–8.08
(m, 1 H, ArH), 8.43 (s, 1 H, OH), 8.68 (s, 1 H, ]CH), 12.31 (s, 1 H, NH);
13
C NMR (DMSO) δ (ppm) = 111.0, 112.5, 121.5, 123.4, 124.2, 126.3,
127.5, 128.2, 128.7, 129.1, 130.4, 130.5, 132.2, 132.8, 133.8, 144.7
(16Car), 154.8 (]CH), 164.8 (C]O). Analysis for C18H13ClN2O2
(324.76) Calculated: C: 66.57%, H: 4.03%, N: 8.63%; Found: C:
66.64%, H: 4.06%, N: 8.57%.
N-[(2-bromophenyl)methylidene]-3-hydroxynaphthalene-2-carbo-
hydrazide (2)
Yield: 92%; m.p.: 244–246 °C. 1H NMR (DMSO-d6) δ
(ppm) = 7.21–7.43 (m, 4 H, ArH), 7.49–7.53 (m, 2 H, ArH), 7.71–7.79
(m, 2 H, ArH), 7.91–7.94 (d, 1 H, ArH, J = 9 Hz), 8.03–8.07 (d, 1 H,
ArH, J = 3 Hz), 8.44 (s, 1 H, OH), 8.82 (s, 1 H, ]CH), 12.27 (s, 1 H,
NH); 13C NMR (DMSO) δ (ppm) = 111.1, 112.2, 121.6, 123.4, 124.2,
124.3, 126.5, 127.2, 127.9, 128.1, 128.7, 133.4, 147.1, 153.8 (16Car),
154.7 (]CH), 164.8 (C]O). Analysis for C18H13BrN2O2 (369.21)
Calculated: C: 58.56%, H: 3.55%, N: 7.59%; Found: C: 58.59%, H:
3.51%, N: 7.64%.
N-[(2-fluorophenyl)methylidene]-3-hydroxynaphthalene-2-carbo-
hydrazide (3)
Yield: 93%; m.p.: 238–240 °C. 1H NMR (DMSO-d6) δ
(ppm) = 7.22–7.40 (m, 5 H, ArH), 7.49–7.57 (m, 2 H, ArH), 7.76–7.79
(d, 1 H, ArH, J = 9 Hz), 7.90–7.93 (d, 1 H, ArH, J = 9 Hz), 7.90–7.96
(m, 1 H, ArH), 8.44 (s, 1 H, OH), 8.70 (s, 1 H, ]CH), 12.13 (s, 1 H, NH);
13
C NMR (DMSO) δ (ppm) = 111.0, 116.4, 116.7, 120.9, 122.1, 122.3,
124.3, 125.5, 126.3, 126.9, 127.2, 128.7, 129.1, 130.6, 136.3, 141.6
1338
Biomedicine & Pharmacotherapy 103 (2018) 1337–1347
(16Car), 154.7 (]CH), 164.5 (C]O). Analysis for C18H13FN2O2
(308.31) Calculated: C: 70.12%, H: 4.25%, N: 9.09%; Found: C:
70.17%, H: 4.28%, N: 9.01%.
2.1.2. Synthesis of 1,3-thiazolidin-4-one derivatives (4–6)
For the synthesis of 1,3-thiazolidin-4-one derivatives (4–6) we ap-
plied the same procedure as we described earlier [13]. 0.01 mol of
appropriate hydrazide-hydrazone of 3-hydroxy-2-naphtoic acid (1–3)
was added to 15 mL of 1,4-dioxane. Subsequently, mercaptoacetic acid
(0.92 g, 0.01 mol) was added dropwise to this solution. The mixture was
stirred for 6 h at room temperature. After that the solvent was removed
under reduced pressure and 15 mL of 10% water solution of sodium
bicarbonate was added to the residue. The formed precipitate was fil-
tered off and re-crystallized from ethanol.
N-[2-(2-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydro-
xynaphthalene-2-carboxamide (4)
Yield: 63%; m.p.: 250–252 °C. 1H NMR (DMSO-d6) δ (ppm) = 3.64
(s, 2 H, CH2), 6.90 (s, 1 H, CH), 7.05–7.07 (m, 1 H, ArH), 7.13–7.15 (m,
2 H, ArH), 7.32–7.35 (m, 3 H, ArH), 7.41–7.44 (m, 1 H, ArH), 7.77–7.79
(m, 2 H, ArH), 7.98–8.00 (d, 1 H, ArH, J = 6 Hz), 8.39 (s, 1 H, OH),
10.91 (s, 1 H, NH); 13C NMR (DMSO) δ (ppm) = 33.3 (CH2), 64.7 (CH),
112.5, 121.5, 123.2, 123.4, 126.7, 126.8, 127.7, 128.8, 129.5, 130.2,
130.4, 131.8, 131.9, 136.7, 139.9, 155.8 (16Car), 162.6 (C]O), 172.3
(C]O). Analysis for C20H15ClN2O3S (398.86) Calculated: C: 60.22%, H:
3.79%, N: 7.02%; Found: C: 60.29%, H: 3.82%, N: 6.97%.
N-[2-(2-bromophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydro-
xynaphthalene-2-carboxamide (5)
Yield: 60%; m.p.: 252–254 °C. 1H NMR (DMSO-d6) δ (ppm) = 3.61
(s, 2 H, CH2), 6.85 (s, 1 H, CH), 7.02–7.03 (m, 1 H, ArH), 7.10–7.12 (m,
1 H, ArH), 7.18–7.20 (m, 1 H, ArH), 7.35–7.37 (m, 2 H, ArH), 7.44–7.48
(m, 2 H, ArH), 7.78–7.80 (m, 2 H, ArH), 7.99-8.01 (d, 1 H, ArH, J
= 6 Hz), 8.42 (s, 1 H, OH), 10.87 (s, 1 H, NH); 13C NMR (DMSO) δ
(ppm) = 33.3 (CH2), 65.8 (CH), 112.5, 121.5, 123.3, 123.4, 126.7,
127.1, 128.8, 128.9, 129.5, 130.2, 130.4, 131.3, 132.8, 136.7, 139.5,
155.9 (16Car), 162.9 (C]O), 172.1 (C]O). Analysis for
C20H15BrN2O3S (443.31) Calculated: C: 54.19%, H: 3.41%, N: 6.32%;
Found: C: 54.10%, H: 3.44%, N: 6.28%.
N-[2-(2-fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydro-
xynaphthalene-2-carboxamide (6)
Yield: 84%; m.p.: 226–228 °C. 1H NMR (DMSO-d6) δ (ppm) = 3.66
(s, 2 H, CH2), 6.55 (s, 1 H, CH), 7.01–7.03 (m, 2 H, ArH), 7.08–7.11 (m,
1 H, ArH), 7.19–7.22 (m, 1 H, ArH), 7.36–7.38 (m, 2 H, ArH), 7.45–7.49
(m, 1 H, ArH), 7.77–7.79 (m, 2 H, ArH), 7.96-7.98 (d, 1 H, ArH, J
= 6 Hz), 8.42 (s, 1 H, OH), 10.87 (s, 1 H, NH); 13C NMR (DMSO) δ
(ppm) = 33.2 (CH2), 64.8 (CH), 112.5, 117.4, 121.5, 123.4, 124.2,
126.7, 127.9, 128.6, 128.8, 129.5, 130.2, 130.4, 132.8, 136.8, 155.6,
160.2 (16Car), 162.1 (C]O), 172.4 (C]O). Analysis for C20H15FN2O3S
(382.41) Calculated: C: 62.82%, H: 3.95%, N: 7.33%; Found: C:
62.78%, H: 3.98%, N: 7.38%.
2.2. In vitro cytotoxicity assays
2.2.1. Cell cultures
This research was performed with the use of tumor cell cultures
supplied by American Type Culture Collection (Manassas, VA, USA), i.e.
HepG2 (hepatocellular carcinoma, human, ATTC®-Number: HB-8065™)
and 769-P (renal cell adenocarcinoma, human, ATCC®-Number: CRL-
1933™). Normal cells were used as reference lines: rat cardiac myo-
blasts (cardiomyocytes) obtained from ATCC - H9c2(2-1) (ATCC®-
Number: CRL-1446™) and green monkey kidney cells (GMK) obtained
from Biomed Serum and Vaccine Production Plant Ltd (Lublin, Poland).
The cell medium for GMK was basic RPMI-1640 (with L-Glutamine,
Hepes and sodium dicarbonate), and for H9c2 it was Dulbecco's
Modified Eagle's Medium (DMEM). The HepG2 cell culture was grown
in Eagle's Minimal Essential Medium (EMEM), while 769-P cell line was
grown in rich-component RPMI-1640 (with L-Glutamine, sodium
Ł. Popiołek et al.
pyruvate, glucose, Hepes and sodium dicarbonate). All the media were
supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin,
100 μg/mL streptomycin and 2.5 μg/mL of amphotericin B. The cell
cultures were grown in 75 cm2 tissue culture flasks (EasYFlasks™
Nunclon™ Δ, Nunc GmbH Wiesbaden, Germany) as monolayer in hu-
midified atmosphere of 5% CO2 at 37 °C in the cell incubator. The
suspensions of H9c2, GMK, 769-P and HepG2 cells were prepared at a
density of 1 × 106 cells/mL and then transferred to 96-well cell culture
plates (SPL Lifescience, Gyeonggi-do, Korea). The prepared plates were
incubated for 24 h in order to achieve cell adhesion to the plates.
2.2.2. Reagents and solvents
In this research we used the following substances and solvents: MTT
(Thiazolyl blue tetrazolium bromide, Sigma-Aldrich, Steinheim,
Germany), DMSO (Dimethyl sulfoxide, Avantor Performance Materials,
Poland S.A., Gliwice, Poland), PBS (Phosphate Buffered Saline, Biomed
Serum and Vaccine Production Plant Ltd, Lublin, Poland). The Eagle's
Minimal Essential Medium (EMEM) and Dulbecco's Modified Eagle's
Medium (DMEM) were obtained from American Type Culture
Collection (Manassas, VA, USA). The basic as well as the rich-compo-
nent RPMI-1640, Fetal Bovine Serum (FBS), antibiotics solutions: pe-
nicillin, streptomycin, amphotericin B, and trypsin solution (Trypsin
0.25% / EDTA 0.02% in PBS w/o Ca, Mg with phenol red) were sup-
plied by PAN-Biotech GmbH, Aidenbach, Germany.
2.2.3. Cell viability assay
Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) test based on DB-ALM Protocol n°17
(ECVAM – European Centre for the Validation of Alternative Methods,
Database Service on Alternative Methods to Animal Experimentation).
The viability is determined in a mitochondrial-dependent reaction
(reduction in mitochondrial dehydrogenase activity) by measurement
of the formazan production from MTT salt. It is expressed as percentage
(%) of control cells. The examined compounds were first dissolved in
DMSO and after that diluted to the required concentration with the
respective cell culture medium. The solutions were prepared ex tempore
and added to the cells in the same volume (100 μl/well) and incubated
for 24 h. After the incubation, 10 μl of MTT solution (5 mg/mL) was
added to each well on a microplate and incubated for 3 h at 37 °C. At
the end of the incubation time the culture medium was carefully re-
moved from each well and 100 μl DMSO was added. The absorbance of
each well was measured at 550 nm using an automated absorbance
microplate reader ELx808IU (Bio-Tek Instruments Inc., USA). The ex-
periments included the determination of IC10, IC25 and IC50 values for
the tested compounds. Subsequently, the cell cultures were incubated in
the presence of the tested compounds at the concentrations of IC10 and
IC25 during 24 h, 48 h and 72 h. All of the parameters mentioned above
were evaluated in the presence of the solvent of the tested derivatives
and there were no significant differences between the control cells and
the solvent-treated cells. All the experiments were performed at least
five times.
2.2.4. Statistical analysis
The results are expressed as mean ± standard deviation (SD). The
statistical significance among the groups was determined by the
Student’s t-test or the UMann-Whitney’s test. The p-value less than 0.05
was chosen as the criterion of statistical significance.
2.3. In vitro antimicrobial assay
The examined compounds 1–3 were in vitro tested for antimicrobial
activity with the use of broth microdilution method according to
European Committee on Antimicrobial Susceptibility Testing (EUCAST)
[14] and Clinical and Laboratory Standards Institute guidelines [15].
This procedure was applied earlier by our group for testing 1,3-thia-
zolidin-4-one derivatives [16–18]. Detailed information concerning
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Biomedicine & Pharmacotherapy 103 (2018) 1337–1347
bacterial and fungal strains used in our in vitro antimicrobial assays is
presented in Supplementary materials.
2.4. In vivo behavioral tests
2.4.1. Animals
The experiments were carried out on adult Swiss mice (20–25 g)
obtained from a licensed breeder (Breeding of Laboratory Animals,
Jacek Kołacz, Poland). The animals were housed in colony cages at
room temperature of 22 ± 1 °C, with free access to tap water and food
(Murigran pellets, Bacutil, Motycz). The animals were maintained on a
normal day-night cycle (light phase 7 a.m.–7 p.m.). After seven days of
acclimatization to standard laboratory conditions, the animals were
divided into groups (8 animals per group) and prepared for the tests.
The experiments were performed between 8 a.m. and 3 p.m. The ex-
perimental protocols and procedures were approved by the ethics
committee (Local Ethics Committee on Animal Experimentation of the
Medical University of Lublin, Poland. The date of this approval: 21th
February 2014).
2.4.2. Substances and solvents
Acetic acid (Sigma-Aldrich, Steinheim, Germany) was used in the
experiments. Before behavioral experiments, all examined compounds
were suspended in a little amount (3–4 drops) of Tween 80 (Sigma, St.
Louis, MO, USA) and then diluted in the saline. All compounds were
administered intraperitoneally (ip) in the maximum doses equivalent to
0.1 of their LD50, in a volume of 0.1 mL/10 g of the body weight. The
control animals received the same volumes of saline with small amount
(3–4 drops) of Tween 80 at the respective time before the test.
2.4.3. Behavioral experiments
The acute toxicity of the studied compounds was assessed in mice,
according to the Up-Down-Up method (guidelines OECD No 425), as
LD50 (95%CL) calculated for mortality within 24 h (Table 5). The
pharmacological experiments were initiated in the doses equivalent to
0.1 of LD50, which were gradually reduced until the disappearance of
the biological activity of the compounds.
The assessment of the safety of tested compounds on the central
nervous system of mice was based on the results of the following be-
havioral tests:
The motor impairment was quantified with the chimney test [19].
In this test, 30 min after the administration of the investigated com-
pounds, mice had to climb up backward in a plastic tube (inner dia-
meter 3 cm, length 25 cm). Mice which were unable to perform this task
within 60 s were considered to display motor impairment. The motor
impairment was quantified as the percentage of animals that failed to
complete the test.
The passive avoidance test was used to evaluate learning and
memory in rodent models of CNS disorders [20]. The apparatus used to
perform this test is divided into bright and dark compartments with a
gate between them. During first day of the test, 30 min after in-
traperitoneally injection of the compound, mice were placed in the
bright compartment. When the animal innately crossed to the black
compartment, it received a mild foot shock. During the initial phase the
animal learned that moving to the dark compartment may have nega-
tive consequences. After 24 h the animal was placed again in the white
compartment and observed for 180 s. The animals which did not go to
the dark part of the apparatus did not display an impaired memory.
The effects of 1,3-thiazolidin-4-one derivatives (4–6) on cognitive
activity were evaluated by the hole board test. The hole board test was
performed according to the procedure in the literature described by
Boissier and Simon [21–23] and Boissier et al. [24]. The animals were
placed on a board (40 × 40 cm) with 16 holes which were symme-
trically distributed in four rows. After 30 min of administration of the
tested substances each animal was placed carefully in the center of the
board and the number of head pokes (head was dipped into the hole)
Ł. Popiołek et al. Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

during 5 min was recorded. The hole board test is used in scientific [26,27]. Both compounds were dissolved in DMSO to a final con-
research to measure anxiety, stress, neophilia and emotionality in ani- centration of 0.2 μg/ml for hydrazide (H) and 10.0 μg/ml for hydrazide-
mals. hydrazone (3). The solutions were kept at 25 °C and the UV–VIS spectra
The antinociceptive activity of the examined compounds was es- were recorded over a period of 0, 0.5, 1, 2, 24, 48 and 72 h. Appropriate
tablished in analgesic test – the writhing test [25]. The writhing test is a UV–VIS spectra of both compounds are presented in Supplementary
chemical analgesic test used to study the peripheral pain induced by materials (Figs. 1S–14S).
intraperitoneally injection of irritant acetic acid (0.6%), as a nocicep-
tive stimulus which induces characteristic writhing episodes (episodes
3.2. In vitro cytotoxicity assays
of retraction of abdomen and stretching of hind limbs). This test was
performed by the injection of acetic acid 30 min after the administra-
The assessment of in vitro cytotoxicity of 1,3-thiazolidin-4-one de-
tion of the compounds and the number of writhing episodes was re-
rivatives (4–6) and their hydrazide-hydrazones (1–3) was performed
corded for a period of 30 min. The analgesic activity of the tested
against human renal cell adenocarcinoma (769-P) and human hepato-
compounds is inferred as the decrease in the frequency of writhings.
cellular carcinoma (HepG2). As normal reference lines, rat cardio-
The analgesic responses were calculated as percent of the inhibition of
myocytes (H9c2) and green monkey kidney cells (GMK) were used. The
the pain reaction.
viability of the aforesaid cells was evaluated by MTT method. The IC50
as well as IC10 and IC25 values for compounds 1–6 (Table 1) were de-
2.4.4. Data analysis termined on the basis of dose-response curves.
The results are expressed as mean ± standard error of the mean
(SEM). The statistical significance among the groups was determined by
3.2.1. In vitro cytotoxicity assays against 769-P cell line
the Student’s t-test or the UMann-Whitney’s test. The p-value less than
Among novel tested derivatives (1–6), compound 1 was the most
0.05 was chosen as the criterion of statistical significance.
toxic towards 769-P and inhibited cancer cell growth with an IC50 value
estimated at 109.5 μM (Table 1). Extending the incubation time to 48 h
3. Results
and 72 h significantly increased the cytotoxic potential of all tested
derivatives (Figs. 1 and 2). The highest decrease in viability (about 93%
3.1. Chemistry
vs. control) was noted after 48 h of incubation for the compound 6 with
these cells at IC25 (35.28 μM). Compound 5 also caused significant and
New 1,3-thiazolidin-4-one derivatives (4–6) were synthesized with
dose-depend increase of cytotoxicity vs. 769-P up to 88% after 48 h
the use of two stage synthesis. Firstly, new hydrazide-hydrazones with
incubation period at IC25 (42.40 μM) (Fig. 2). After 48 h and 72 h of
3-hydroxy-2-naphthoic moiety (1–3) were synthesized with the use of
incubation time of human renal adenocarcinoma cells (769-P) with
condensation reaction of 3-hydroxy-2-naphthoic acid hydrazide (H)
compound 2 at IC10 (16.90 μM) and at IC25 (45.05 μM), a significant
with appropriate substituted aromatic aldehydes. Then, newly obtained
reduction of cell viability was reported, i.e. up to 48% and 39% ac-
hydrazide-hydrazones (1–3) were subjected to cyclization reaction with
cording to the incubation time and according to the inhibitory con-
mercaptoacetic acid in the presence of 1,4-dioxane which led to new
centration used. The dose-dependent and the time-dependent increase
1,3-thiazolidin-4-one derivatives (4–6). The chemical structure of all
obtained compounds (1–6) was confirmed on the basis of spectral
Table 1
studies (1H NMR and 13C NMR) as well as elemental analysis. Synthetic The IC50 determination for newly synthesized 1,3-thiazolidin-4-ones (4–6) and
reactions performed in this research are shown in Scheme 1. their hydrazide-hydrazones (1–3) after 24 h of incubation.
It is worth to mention that synthesized hydrazide-hydrazones (1–3)
No of compound IC50 (μM)
and 1,3-thiazolidin-4-one derivatives (4–6) with 3-hydroxy-2-naph-
thoic moiety are new and their synthesis, spectral characterization, in 769-P HepG2 H9c2 GMK
vitro and in vivo properties have not been reported in the scientific lit-
erature so far. 1 109.50 8.75 114.35 117.50
2 230.70 445.10 nd 577.30
3 221.40 135.70 110.46 54.79
3.1.1. Stability study 4 152.97 13.33 69.85 114.43
The stability of 3-hydroxy-2-naphthoic acid hydrazide (H) and 5 237.99 16.71 119.30 127.41
synthesized hydrazide-hydrazone: N-[(2-fluorophenyl)methylidene]-3- 6 164.00 9.54 32.46 100.00

hydroxynaphthalene-2-carbohydrazide (3) in DMSO was monitored

nd – Not determined.
spectrophotometrically according to procedure reported earlier

Scheme 1. Synthesis of new hydrazide-hydrazones and 1,3-thiazolidin-4-one derivatives.

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Ł. Popiołek et al. Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

Fig. 1. The effect of 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) at a concentration of IC10 on the 769-P cells viability in the MTT test. Data is
presented as percentage of the cell viability ± SD. *p < 0.05 ; **p < 0.01 ; ***p < 0.001 vs. control cells.

of cytotoxicity was also noted in the case of compound 3, when applied
at IC10 (6.26 μM) to 38% and at IC25 (23.85 μM) to 50% (Figs. 1 and 2).
3.2.2. In vitro cytotoxicity assays against HepG2 cell line
The lowest value of 50%-inhibitory concentration was noted for
compound 1 (8.75 μM) and a similar value for compound 6 (9.54 μM) in
human hepatocellular carcinoma cell line (HepG2) (Table 1). The
highest reduction of cell viability was observed for 25%-inhibitory
concentrations of the compound 2 (44.0 μM) and 3 (17.30 μM) com-
pared with control cells after 48 h of the incubation period. The via-
bility was about 60% and 50% lower, respectively (Table 2). In addi-
tion, during the same time of incubation, the aforementioned
substances applied at IC10 evoked the increase of cytotoxic effect in
HepG2 cells (45% growth inhibition for the compound 2 at 10.97 μM
and for the compound 3 at 5.03 μM vs. control) (Table 2).

Fig. 2. The effect of 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) at a concentration of IC25 on the 769-P cells viability in the MTT test. Data is
presented as percentage of the cell viability ± SD. *p < 0.05 ; **p < 0.01 ; ***p < 0.001 vs. control cells.

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Ł. Popiołek et al. Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

Table 2
The effect of newly synthesized 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) on the viability of HepG2 cells after 24 h, 48 h or 72 h of
incubation.
Compound No Concentration The viability of the cells after incubation (%)

24 h 48 h 72 h

1 IC10 91.21 ± 4.32 107.52 ± 3.31 69.26 ± 21.42

IC25 79.22 ± 4.54** 101.55 ± 4.21 89.96 ± 18.59
2 IC10 90.53 ± 2.88 53.02 ± 5.93*** 74.66 ± 4.98**
IC25 75.15 ± 6.15** 43.07 ± 3.18*** 60.27 ± 7.06***
3 IC10 92.03 ± 5.98 54.76 ± 2.03*** 87.71 ± 9.19
IC25 77.25 ± 6.15** 49.18 ± 2.16*** 71.41 ± 5.94**
4 IC10 90.83 ± 4.46 105.29 ± 4.02 83.83 ± 18.56
IC25 79.38 ± 9.80** 90.02 ± 4.40 109.30 ± 6.90
5 IC10 92.51 ± 7.08 106.49 ± 3.46 115.54 ± 5.83
IC25 78.11 ± 8.34** 102.99 ± 3.49 115.40 ± 6.88
6 IC10 94.22 ± 4.75 98.01 ± 3.48 99.96 ± 13.14
IC25 74.85 ± 5.35** 104.12 ± 3.71 73.80 ± 18.67 **

Data is presented as percentage of the cell viability ± SD.

*p < 0.05.
** p < 0.01.
*** p < 0.001 vs. control cells.

Table 3
The effect of newly synthesized 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) on the viability of H9c2 cells after 24 h, 48 h or 72 h of incubation.
Compound No Concentration The viability of the cells after incubation (%)

24 h 48 h 72 h

***
1 IC10 89.17 ± 5.32 51.95 ± 4.24 96.32 ± 8.51
IC25 75.88 ± 4.54** 44.79 ± 1.99*** 66.71 ± 4.41***
3 IC10 92.03 ± 5.98 79.03 ± 28.26 83.35 ± 11.34
IC25 76.25 ± 4.15** 57.48 ± 4.75*** 50.34 ± 15.28***
4 IC10 89.03 ± 5.98 82.44 ± 10.57 80.58 ± 8.68**
IC25 74.67 ± 7.29** 37.54 ± 4.29*** 45.09 ± 4.94***
5 IC10 89.18 ± 7.11 32.78 ± 4.97*** 46.79 ± 6.25***
IC25 73.35 ± 5.58** 26.94 ± 2.49*** 39.87 ± 3.81***
6 IC10 92.53 ± 6.44 97.20 ± 9.25 93.10 ± 6.63
IC25 79.25 ± 7.08** 95.79 ± 6.47 88.60 ± 11.40

Data is presented as percentage of the cell viability ± SD.

*p < 0.05.
** p < 0.01.
*** p < 0.001 vs. control cells.

3.2.3. In vitro cytotoxicity assays against H9c2 cell line
The cardiomyocytes seemed to be the most sensitive to toxic effect
of compound 6. It caused the 50%-inhibition of cell viability at
32.46 μM (Table 1). However, the extending of the incubation time to
48 h and 72 h caused no significant undesirable changes in cell viability
vs. control. The viability was even higher than expected at concentra-
tions used (Table 3). Among all tested derivatives (1–6), the compound
5 at IC25 (0.768 μM) caused the most significant decrease (almost 75%
vs. control) in cell viability after 48 h (Table 3).
3.3. In vitro antimicrobial assays
The results of our antimicrobial activity study indicated that newly
synthesized hydrazide-hydrazones (1–3) had no antibacterial activity
against Gram-positive and Gram-negative bacterial strains. To the
contrary the compounds 1 and 3 showed some bioactivity against
Candida spp. ATCC strains (Table 4). The lowest MIC values were
shown by compound 3 against Candida albicans ATCC 2091 and Can-
dida parapsilosis ATCC 22019 (MIC = 500 μg/mL, respectively). Com-
pound 2 had no inhibitory effect against Candida spp. (Table 4).

3.2.4. In vitro cytotoxicity assays against GMK cells
Compound 3 was the most toxic towards GMK cells and inhibited
cell growth with an IC50 value estimated at 54.79 μM. At the same time,
these normal cells were the most resistant to the antiproliferative po-
tential of compound 2 (IC50 = 577.3 μM) (Table 1). In the case of tested
derivatives (1–6), there were no significant decreases of cell viability
after 24 h, 48 h and 72 h of incubation in comparison with the control
cells. In some cases, significant reductions in viability were noted,
however with regard to the inhibitory concentration used (Figs. 3 and
4). The highest rise of cytotoxicity was detected for compound 1 in-
cubated with GMK for 48 h. This compound reduced the cell viability by
approximately 40%. Similar results were obtained for this compound at
both concentrations, IC10 (9.19 nM) and IC25 (0.12 μM) (Figs. 3 and 4).
3.4. In vivo behavioral tests
3.4.1. The influence of the synthesized compounds on CNS of mice
The 1,3-thiazolidin-4-one derivatives (4–6) were characterized by
low toxicity, which reflected the LD50 doses (all the compounds were
1000 mg/kg ip, Table 5). In addition, the compounds 4–6 had a slight
effect on the CNS of animals, which confirmed their safety. All tested
compounds were devoid of neurotoxicity as they did not impair the
mouse motor coordination (at highest doses). This is very important
from the pharmacological point of view (Table 6). Additionally, none of
the tested compounds administered at the maximal dose (0.1 of their
LD50, Table 6) affected the cognitive activity of mice in hole-board test
and their memory in passive avoidance test. Furthermore, derivative 4

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Ł. Popiołek et al. Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

Fig. 3. The effect of 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) at a concentration of IC10 on the GMK cells viability in the MTT test. Data is
presented as percentage of the cell viability ± SD. *p < 0.05 ; **p < 0.01 ; ***p < 0.001 vs. control cells.

administered at the dose of 100 mg/kg significantly increased cognitive

Table 4
activity of mice compared with the control group. Among the tested
The activity data of the tested compounds (1–3) expressed as MIC (MFC) [μg/
compounds, N-[2-(2-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hy- mL] against the reference strains of fungi.
droxynaphthalene-2-carboxamide (4) and N-[2-(2-fluorophenyl)-4-oxo-
1,3-thiazolidin-3-yl]-3-hydroxynaphthalene-2-carboxamide (6) ex- Species MIC (MFC) [μg/mL] of the tested compounds FLU

hibited highest biological activity. 1 2 3

Candida albicans ATCC 1000 (> 1000) – 500 (> 1000) 0.244
3.4.2. The analgesic activity of 1,3-thiazolidine-4-one derivatives 2091
Candida albicans ATCC 1000 (> 1000) – 1000 (> 1000) 0.976
The writhing test confirmed the biological activity of the tested
10231
compounds (4-6). Derivatives: N-[2-(2-chlorophenyl)-4-oxo-1,3-thia- Candida parapsilosis ATCC – – 500 (> 1000) 1.953
zolidin-3-yl]-3-hydroxynaphthalene-2-carboxamide (4) and N-[2-(2- 22019,019
fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydroxynaphthalene-2-car-
boxamide (6) significantly reduced the number of writhing episodes in The standard compound – fluconazole (FLU) was used as positive control for
fungi.
mice induced by intraperitoneal injection of 0.6% acetic acid (Table 7).
Compound 6 was the most interesting because it had the strongest
analgesic effect over a wide range of doses.

Fig. 4. The effect of 1,3-thiazolidin-4-ones (4–6) and their hydrazide-hydrazones (1–3) at a concentration of IC25 on the GMK cells viability in the MTT test. Data is
presented as percentage of the cell viability ± SD. *p < 0.05 ; **p < 0.01 ; ***p < 0.001 vs. control cells.

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Ł. Popiołek et al. Biomedicine & Pharmacotherapy 103 (2018) 1337–1347

Table 5 ]CH group was observed at δ 154.8 ppm and for carbonyl group (C]
The LD50 values and start doses of the compounds 4–6 used in the behavioral O) around δ 164 ppm.
tests. New 1,3-thiazolidin-4-one derivatives (4–6) in 1H NMR spectra
Compound No LD50 (mg/kg The start dose of the compounds used in the showed signals for CH, CH2 and NH groups in the range of δ
ip) experiments (mg/kg ip) 6.55–6.90 ppm, δ 3.61–3.64 ppm, and δ 10.87–10.91 ppm, respectively.
The 13C NMR spectra also confirmed the occurrence of CH, CH2 and
4 1000 100
carbonyl (C]O) groups in this class of compounds.
5 1000 100
6 1000 100 The presence of the signals above mentioned confirmed correct
cyclization reaction of hydrazide-hydrazones (1–3) to the 2,3-dis-
ubstituted 1,3-thiazolidin-4-one system (4–6).
Table 6
The effect of the compounds 4–6 on the results of the chimney, the passive 4.1.1. Stability study
avoidance and the hole board test in mice. On the basis of registered UV–VIS spectra, we revealed that hy-
Compound No Dose Chimney test Passive avoidance Hole-board test drazide-hydrazone (3) as well as its hydrazide (H) showed a decrease in
[mg/ [s] test [s] [the average intensity of the absorption bands after 24 h–72 h, what may confirm the
kg ip] number of head- hydrolysis or degradation of these compounds.
dipping]

Control group – 8.5 ± 1.0 163.3 ± 16.7 54.3 ± 2.9

4.2. In vitro cytotoxicity assays
4 100 12.0 ± 1.5 164.5 ± 13.1 69.4 ± 3.8*
50 – – 58.3 ± 3.1 The 1,3-thiazolidin-4-one derivatives show anticancer activity
5 100 13.4 ± 3.7 165.3 ± 14.75 55.4 ± 6.0 [28,29]. Numerous studies confirmed that 2,3-disubstituted 1,3-thia-
6 100 10.9 ± 4.3 180 ± 0.0 60.3 ± 6.6
zolidin-4-ones were reported for antiproliferative activity, i.e. in human
* p < 0.05 in comparison with the control group ; The tested compounds and colon adenocarcinoma cells (HT29), human gastric cancer cell line,
saline were administered ip 30 min before the test. Each experimental group human colon cancer cell lines, and sarcoma-derived cells [30,31]. The
consisted of 8 animals. Data is presented as the Mean ± SEM. The statistical 2,3,5-trisubstituted derivatives were revealed to induce cell growth
significance among the groups was determined by the Student’s t-test or the arrest in HT29 and significant cytotoxicity towards human lung and
UMann-Whitney’s test. human breast cancer cultures [32]. The 2,3-disubstituted 1,3-thiazo-
lidin-4-one derivatives showed antiviability potential against leukemic
Table 7 cell lines [28]. In turn, 2-phenylimino-3-alkyl-1,3-thiazolidin-4-one
The effect of the compounds 4–6 on acetic acid-induced abdominal writhing in derivatives restrained the proliferation of HT29 cells, characterized by
mice. CDK1/cyclin B inhibition. This effect was achieved by the blocking of
cell progression at the G2/M phase border and induction of apoptosis
Compound No Dose [mg/ Writhing test [number of Inhibition [%]1
kg ip] writhing episodes] [6]. It was also reported that 5-benzylidene-1,3-thiazolidin-4-one de-
rivatives demonstrated significant antitumor potential with various
Control group 0.6% acetic 24.6 ± 4.1 – biotargets and mechanisms, such as sphingosine kinase (SK), JNK sti-
acid
mulating phosphatase-1 (JSP-1) or non-membrane protein tyrosine
4 100 10.5 ± 3.2* 57.3*
50 18.0 ± 4.4 26.8
phosphatase (SHP-2) [6,33]. However, it is worth to emphasize that
5 100 19.4 ± 3.2 21.1 there is no available scientific data focused on the effect of the 1,3-
Control group 0.6% acetic 30.6 ± 3.4 – thiazolidin-4-ones on the growth of human cancer lines, like 769-P or
acid HepG2. In turn, numerous hydrazide-hydrazone analogues showed
6 100 13.5 ± 4.0** 55.8**
significant human cancer cell growth inhibition of the following tumor
50 15.1 ± 7.4* 50.6*
25 26.1 ± 4.7 29.4 lines: lung (A549), liver (HepG2), ovarian (SK-OV-3), breast (MCF-7),
cervical (HeLa), skin (SK-MEL-2), colon (HCT15), gastric (Bc G-823)
1
The percent of inhibition obtained by the comparison with the control and pancreatic (MIA-PaCa-2) [34–36]. The facts mentioned above en-
group. couraged us to study the antiproliferative potential of 1,3-thiazolidin-4-
* p < 0.05. ones (4–6) and their hydrazide-hydrazones (1–3) towards renal cell
** p < 0.001 in the comparison of writhing episodes with the control group adenocarcinoma (769-P) and hepatocellular carcinoma (HepG2). It is
; The tested compounds and acetic acid were administered ip 30 min before the generally known that the overall survival of the patients with hepato-
test. The values represent Mean ± SEM (n = 8 per group). Data was analyzed cellular carcinoma (HCC) and also with renal cell carcinoma (RCC)
by the Student’s t-test or the UMann-Whitney’s test. remains poor and available cytostatic agents are insufficient [37,38].
Therefore, there is an urgent need to discover more effective as well as
4. Discussion safe anticancer drugs.
The MTT cell viability assay of newly synthesized 1,3-thiazolidin-4-
4.1. Chemistry ones (4–6) and their hydrazide-hydrazones (1–3) was performed on
human cancer cell lines (renal cell adenocarcinoma 769-P and hepa-
The route for the synthesis of the compounds enumerated in the title tocellular carcinoma HepG2) and normal cell lines (H9c2, GMK) as
was based on our previous reports concerning hydrazide-hydrazones references. The research indicated that compound 1 was the most
and 1,3-thiazolidin-4-ones [12,13]. Successful synthesis of new hy- promising with higher IC50 values estimated for normal cells and lower
drazide-hydrazones (1-3) and 1,3-thiazolidin-4-one derivatives (4-6) for cancer cells, in particular HepG2 (8.75 μM). Unfortunately, pro-
containing 3-hydroxy-2-naphtoic moiety performed in this research was longed incubation revealed undesirable decreases in cell viability of
confirmed on the basis of spectral (1H NMR and 13C NMR) and ele- H9c2 up to 44% after 48 h of incubation with this derivative at IC25
mental analysis. (17.15 μM). Similar reductions by about 40% of the cell growth were
In the 1H NMR spectra of hydrazide-hydrazones (1-3) we found two observed also in GMK cells after the same time of incubation when the
typical singlet signals, characteristic for this group of compounds, compound 1 was applied at both concentrations.
corresponding to ]CH and NH groups at δ 8.68–8.82 ppm and δ Among 1,3-thiazolidin-4-ones (4–6) compounds 4 and 5 were cy-
12.13–12.31 ppm, respectively. At the same time in 13C NMR signal for totoxic mainly against 769-P. In HepG2 cancer cell line, the estimated

1344
Ł. Popiołek et al.
IC50 values (13.33 μM and 16.71 μM, respectively) were lower than for
normal cells but the extending of incubation period did not lead to the
enhancement of cytotoxic effect. In particular, derivative 4 caused very
significant decline of 769-P cell viability up to 40% after 48 h, when it
was applied at IC25 (27.45 μM). What is important, in the case of this
derivative neither the enhancement of the dose applied nor the
prolonging of the incubation time led to the increase of cytotoxic effect
towards GMK cells. However, it significantly reduced the cell growth of
H9c2 at IC25 (3.69 μM). Similarly, the compound 5 at IC25 (42.4 μM)
caused even higher decrease of the cell viability estimated at 88% in
769-P after 48 h of incubation. Unfortunately, it had also significant
cytotoxic effect towards H9c2 at IC10 (6.6 μM) and IC25 (19.55 μM) after
48 h and 72 h. While the reduction of GMK cells growth generally re-
ferred to the inhibitory concentration used.
In turn, compound 2, with IC50 values higher for normal cells than
for cancer cells, was characterized by moderate selectivity. It showed
time-dependent and dose-dependent decreases in the viability of cancer
769-P cell line up to 39%. The highest cytotoxicity for HepG2 cancer
line was estimated at 67%. At the same time, the reduction of the cell
growth noted in GMK cells resulted from the inhibitory concentration
used.
The highest selectivity was shown by the compound 6. It was ap-
plied at the concentration of IC25 to the cancer 769-P cell line, de-
creased significantly the cell viability by approximately 93% after 48 h
and 80% after 72 h of the incubation period, when compared with the
control cells. These were the highest recorded declines of viability,
therefore it may be suggested that fluorine substituent is essential for
the cytotoxicity of the tested derivatives. The time of incubation had no
impact on its cytotoxic effect towards HepG2 cells. During 48 h and
72 h of incubation with compound 6 there was no significant decreases
in the viability of the normal cells (H9c2, GMK) or the decrease was in
accordance with inhibitory concentration used, what confirmed high
selectivity and safety of this derivative. Its hydrazide-hydrazone (3) was
characterized by less selectivity. However, it significantly inhibited the
growth of both cancer lines tested. In the case of 769-P line the com-
pound 3 caused significant reduction in the cell viability in a dose-de-
pendent and time-dependent manner up to 50% when compared to the
untreated cells. The similar growth inhibition was observed in HepG2
cells when incubated for 48 h with this derivative at both concentra-
tions, IC10 (5.03 μM) and IC25 (17.30 μM). Our study confirmed that
hydrazide-hydrazone moiety seemed to be much favourable for cyto-
toxic activity, in particular against HepG2 cells, which was reported by
He et al. earlier [35]. The hydrazide-hydrazone moiety as pharmaco-
phore played a significant antitumor activity in many antitumor agents
[35,36]. It was revealed that the derivative with this pharmacophore
and also with the fluoro substituent induced cell apoptosis through S
cell-cycle arrest in HepG2 cells [35]. Unfortunately, the anti-
proliferative activity of compound 3 also concerned H9c2 cells when it
was applied at IC25 (12.67 μM). At the same time, the viability of GMK
cells remained unchanged even after 72 h of the incubation with this
hydrazide-hydrazone at IC25 (2.86 μM).
The plausible cellular mechanism may be associated with triggering
apoptotic cell death. The mitochondria play a pivotal role in the in-
itiation and amplification of most apoptotic pathways. The mitochon-
drial membrane permeability increases, when these organelle are sti-
mulated by the apoptotic signals [30,39]. Our results of MTT assay
revealed the impairment of mitochondrial enzymes which are involved
in tetrazolic salt metabolism. It could suggest the metabolic dysfunction
and lower mitochondrial reserve capacity in the cancer cells after the
exposure to novel molecules tested. On the other hand, it may be hy-
pothesized that the inhibition of human cancer cell growth observed in
769-P and HepG2 after the exposure to 1,3-thiazolidin-4-one deriva-
tives could be associated with the impact on cyclooxygenase-2 (COX-2)
which seems to be even more interesting. Many types of cancer have
been correlated with the increased levels of COX-2, the enzyme that
plays a key role in apoptosis, angiogenesis and metastasis [32,40]. This
1345
Biomedicine & Pharmacotherapy 103 (2018) 1337–1347
isoform of cyclooxygenase has been proposed as an important cellular
factor associated also with carcinogenesis in hepatocellular carcinoma
and renal cell carcinoma [41–43]. The COX-2 has been proved to in-
fluence the tumor initiation, its progression and invasion. Therefore,
COX-2 inhibitors are suggested to possess chemotherapeutic properties
in established and late-stage malignancies [40]. The upregulation of
this protein in transformed cells and in tumors led to the increased
production of prostaglandins, especially PGE2 [32,40]. The results of
recent in vitro studies in five cancer lines with overexpression of COX-2
and increased prostaglandin production confirmed that novel diphe-
nylthiazole substituted 1,3-thiazolidin-4-one derivatives exhibited good
antitumor activity via inhibiting COX-2 enzyme [44]. However, further
detail studies should be conducted to verify aforementioned hy-
potheses.
4.3. In vitro antimicrobial assays
Newly synthesized compounds (1-3) had no antibacterial activity
but displayed weak activity against Candida spp. (MIC = 500–1000 μg/
mL). The reason for the lack of bactericidal or bacteriostatic activity of
this group of hydrazide-hydrazones (1-3) may be connected with the
fact that these compounds were derived from 3-hydroxy-2-naphthoic
acid hydrazide (H) which itself did not display substantial antibacterial
activity.
Similar hydrazide-hydrazones, synthesized in our previous research,
but obtained from different carboxylic acids possessed interesting and
significant antimicrobial properties [12].
4.4. In vivo behavioral tests
Compounds 4-6 had a slight effect on the CNS of animals, which
confirms their safety. None of administered compounds at the maximal
dose (0.1 of their LD50, Table 6) disturbed the motor coordination,
cognitive activity or the memory of mice, which emphasizes the un-
iqueness of these derivatives. Among the tested compounds, the N-[2-
(2-chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydroxynaphthalene-2-
carboxamide (4) and N-[2-(2-fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-
3-hydroxynaphthalene-2-carboxamide (6) exhibited the highest biolo-
gical activity. The analgesic activities of the novel compounds 4–6 were
determined in mice using well known "writhing" test, which is fre-
quently used to test for peripherally acting drugs.
In response to the pain caused by the intraperitoneal injection of
0.6% acetic acid come to liberating endogenous substances as well as
some other pain mediators such as arachidonic acid metabolites via
cyclooxygenases, such as prostaglandins.
The observed reduction in the number of the episodes of stretching
of the hind limbs after administration of the investigated N-[2-(2-
chlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-hydroxynaphthalene-2-car-
boxamide (4) and N-[2-(2-fluorophenyl)-4-oxo-1,3-thiazolidin-3-yl]-3-
hydroxynaphthalene-2-carboxamide (6) derivatives can be related to
the inhibition of prostaglandin synthesis, which is characteristic for the
mechanism of action of non-steroidal anti-inflammatory drugs
(NSAIDs) (Table 7) [45–47]. The confirmation of the described me-
chanism may be found in the studies by Jain et al. [2], who considered
that 1,3-thiazolidin-4-one derivatives which have chlorine atom in the
phenyl ring in the second position may show anti-inflammatory action.
Additionally Uchoa’s et al. [48] studies showed that 1,3-thiazolidin-4-
one derivatives which contain chlorine atom in the phenyl ring in the
fourth position can effectively inhibit inflammation (in 67.2%), at-
taining peak plasma concentrations between 0.5 and 1 h.
5. Conclusions
In conclusion, in this research we obtained and identified on the
basis of spectral analysis new hydrazide-hydrazones (1-3) and 1,3-
thiazolidin-4-one derivatives (4-6) with 3-hydroxy-2-naphthoic moiety.
Ł. Popiołek et al.
All of synthesized compounds were subjected to in vitro and in vivo
studies.
In brief, we observed on the basis of conducted assays that 1,3-
thiazolidin-4-one derivative (6) possessed high and selective cytotoxi-
city towards renal adenocarcinoma cells (769-P), whereas the growth of
normal cells (H9c2, GMK) remained unaffected or slightly decreased
but with regard to the IC used. In addition to this, its hydrazide-hy-
drazone (3) had significant cytotoxic potential towards both cancer cell
lines tested (renal cell adenocarcinoma and hepatocellular carcinoma),
however with less selectivity. Similarly, compound 2 had higher IC50
values for normal cells than for cancer cells, which seems to be very
promising. In addition to this, compound 2 had no impact on the
growth of GMK. Additionally, newly synthesized hydrazide-hydrazones
1 and 3 showed some antifungal activity against reference strains of
Candida spp.
The preliminary screening of 1,3-thiazolidin-4-one derivatives (4–6)
proved that synthesized compounds are safe and not toxic to CNS of
mice. In addition, compound 6 was found to possess significant an-
algesic activity. This potential may be associated with the inhibition of
COX-2-controlled production of prostaglandins. It should be empha-
sized that in human cancer cell lines aforementioned molecule (6) also
exhibited the strongest and the most selective antiproliferative activity.
It is generally known that in cancer treatment the inhibition of COX-2
mediated signalling pathway is one of the possible molecular targets for
novel molecules and therapeutic strategies.
The results obtained in our in vitro and in vivo studies convince us
that 3-hydroxy-2-naphthoic scaffold may be regarded as promising
moiety for further development of safe analgesic and cytotoxic agents
against human cancer cells.
Conflict of interests
Authors declare no conflicts of interests.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2018.04.163.
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