Synthesis, cytotoxicity, and pro-apoptosis activity of etodolac hydrazide derivatives as anticancer agents

Article abstract of DOI:10.1002/ardp.201200449

Etodolac hydrazide and a novel series of etodolac hydrazide-hydrazones 3-15 and etodolac 4-thiazolidinones 16-26 were synthesized in this study. The structures of the new compounds were determined by spectral (FT-IR, ¹H NMR, ¹³C NMR, HREI-MS) methods. Some selected compounds were determined at one dose toward the full panel of 60 human cancer cell lines by the National Cancer Institute (NCI, Bethesda, USA). 2-(1,8-Diethyl-1,3,4,9- tetrahydropyrano[3,4-b]indole-1-yl)acetic acid[(4-chlorophenyl)methylene] hydrazide 9 demonstrated the most marked effect on the prostate cancer cell line PC-3, with 58.24% growth inhibition at 10^-5 M (10 μM). Using the MTT colorimetric method, compound 9 was evaluated in vitro against the prostate cell line PC-3 and the rat fibroblast cell line L-929, for cell viability and growth inhibition at different doses. Compound 9 exhibited anticancer activity with an IC₅₀ value of 54 μM (22.842 μg/mL) against the PC-3 cells and did not display any cytotoxicity toward the L-929 rat fibroblasts, compared to etodolac. In addition, this compound was evaluated for caspase-3 and Bcl-2 activation in the apoptosis pathway, which plays a key role in the treatment of cancer. Etodolac hydrazide and a novel series of etodolac hydrazide-hydrazones 3-15 and etodolac 4-thiazolidinones 16-26 were synthesized. 2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetic acid[(4-chlorophenyl)methylene]hydrazide 9 demonstrated the most marked cell viability and growth inhibition effects. It was evaluated for caspase-3 and Bcl-2 activation in the apoptosis pathway. Copyright

Full text of DOI:10.1002/ardp.201200449

Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
367
Full Paper
Synthesis, Cytotoxicity, and Pro-Apoptosis Activity of
Etodolac Hydrazide Derivatives as Anticancer Agents
1
Pelin C¸ ıkla , Derya Ozsavcı , Ozlem Bingol-Ozakpınar , Azize S¸ener , Ozge C¸ evik
2
2
2
2,3
¨
¨
¨
¨
¨
,
4
4
5
1
¨
˘
Suna Ozbas¸-Turan , Ju¨lide Akbuga , Fikrettin S¸ahin , and S¸. Gu¨niz Ku¨c¸u¨kgu¨zel
1
˙
Department of Pharmaceutical Chemistry, Marmara University, Istanbul, Turkey
2
˙
Department of Biochemistry, Marmara University, Istanbul, Turkey
3 Department of Biochemistry, Cumhuriyet University, Sivas, Turkey
4
˙
Department of Pharmaceutical Biotechnology, Marmara University, Istanbul, Turkey
˙
Department of Genetics and Bioengineering, Yeditepe University, Istanbul, Turkey
5
Etodolac hydrazide and a novel series of etodolac hydrazide-hydrazones 3–15 and etodolac
4-thiazolidinones 16–26 were synthesized in this study. The structures of the new compounds
were determined by spectral (FT-IR, 1H NMR, 13C NMR, HREI-MS) methods. Some selected
compounds were determined at one dose toward the full panel of 60 human cancer cell lines by
the National Cancer Institute (NCI, Bethesda, USA). 2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-
b]indole-1-yl)acetic acid[(4-chlorophenyl)methylene]hydrazide 9 demonstrated the most marked
effect on the prostate cancer cell line PC-3, with 58.24% growth inhibition at 10ꢀ5 M (10 mM).
Using the MTT colorimetric method, compound 9 was evaluated in vitro against the prostate cell
line PC-3 and the rat fibroblast cell line L-929, for cell viability and growth inhibition at different doses.
Compound 9 exhibited anticancer activity with an IC50 value of 54 mM (22.842 mg/mL) against the PC-3
cells and did not display any cytotoxicity toward the L-929 rat fibroblasts, compared to etodolac. In
addition, this compound was evaluated for caspase-3 and Bcl-2 activation in the apoptosis pathway,
which plays a key role in the treatment of cancer.
Keywords: Apoptosis / Etodolac / Hydrazide-hydrazone / PC-3 prostate cancer cell line / 4-Thiazolidinone
Received: November 28, 2012; Revised: March 6, 2013; Accepted: March 8, 2013
DOI 10.1002/ardp.201200449
Additional supporting information may be found in the online version of this article at the publisher’s
:
web-site.
Introduction
effects on cyclooxygenase-2 (COX-2) activation. Its mechanism
of action is the inhibition of COX, leading to a reduction in
the synthesis of prostaglandins and other mediators of
inflammation and pain. Prostaglandins are thought to play
an important role in the proliferation of prostate cancer
and are highly expressed in prostate cancer tissues.
Epidemiological studies, clinical observations, and animal
model studies suggest that NSAIDs have the potential to
protect against carcinogenesis due to their inhibitory effect
on COX-2 [1–3]. In recent years, anti-cancer effects of NSAIDs
Etodolac
((R,S)
2-[1,8-diethyl-1,3,4-tetrahydrapyrano(3,4-
b)indole-1-yl]acetic acid) is a nonsteroidal anti-inflammatory
drug (NSAID) with analgesic and antipyretic properties.
The U.S. Food and Drug Administration approved etodolac
for the treatment of inflammation and pain caused by osteo-
arthritis and rheumatoid arthritis. Etodolac has inhibitory
through
a COX-2-independent mechanism have been
Correspondence: Prof. S¸. Gu¨niz Ku¨c¸u¨kgu¨zel, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University,
˙
Haydarpas¸a, 34668 Istanbul, Turkey.
E-mail: gkucukguzel@marmara.edu.tr
Fax: þ90-216-3452952
revealed. COX-2 inhibitors such as etodolac suppress prolifer-
ation, induce apoptosis in prostate cancer cells and have no
effect on normal prostate stromal cells [4, 5]. Recently, it has
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368
P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
been reported that etodolac has antitumor activity on many
types of cancer, such as urogenital system cancers [4, 6],
Burkitt’s lymphoma [7], multiple myeloma, chronic lympho-
cytic leukemia, and prostate cancer [8–10].
using a Dean–Stark water separator. Purification of the syn-
thesized compounds in this study was confirmed by thin-
layer chromatography (TLC) and microanalysis. The struc-
tures of these compounds synthesized from etodolac were
identified by the help of elemental analysis and FT-IR,
1H NMR, and HR-MS (compounds 16–26) spectral data.
Etodolac methyl ester has previously been reported in the
literature [28, 29]; however, we synthesized this compound
using a different method in this study. Etodolac hydrazide 2
–
Hydrazones possessing an azomethine (–NHN CH–) pro-
–
ton constitute an important class of compounds for new drug
development. Therefore, many researchers have synthesized
these compounds as target structures and evaluated their
biological activities [11]. The search for more effective and
less toxic anticancer drugs led to the discovery of hydrazide-
hydrazones including new compounds having anticancer
activity [12]. In previous studies, it has been reported that
hydrazide-hydrazones possess antitumoral activity [13–20].
There is also a report on the discovery of a hydrazone deriva-
tive as an apoptosis inducer that caused a high degree of
growth inhibition in A549 lung cancer cells [21].
1
was first reported in this study. In the H NMR spectrum of
2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)aceto-
hydrazide 2, singlet signals derived from the hydrazide struc-
ture appeared at 4.27 ppm (–NHNH2) and 8.94 ppm
(–NHNH2), showing the integration of two protons and one
proton, respectively.
Etodolac hydrazide-hydrazones were characterized by IR
ꢀ1
–
–
–
Another important biological scaffold is given by 4-thiazo-
lidinones, which are used in the field of medicinal chemistry
and possess a wide range of promising biological activities
[22]. The 4-thiazolidinone derivatives have demonstrated sig-
nificant anticancer activity [23–27].
spectra displaying C O bands at 1639–1667 cm and C N
–
bands at 1601–1630 cmꢀ1 [19, 30]. The 1H NMR data were also
in agreement with the formation of hydrazide-hydrazone.
The signals appearing at 7.92–8.46 ppm in the 1H NMR spec-
–
tra were attributed to the –N CH proton, and the proton loss
–
at 4.27 ppm was attributed to –CO–NH–NH2 [31]. According
These facts and our interest in the synthesis of newer
anticancer compounds prompted us to undertake the syn-
thesis of novel hydrazide-hydrazones and their subsequent
conversion into 4-thiazolidinone derivatives starting from
etodolac and to evaluate their anticancer potential. These
observations guided us in the development of new etodolac
hydrazide-hydrazones and 4-thiazolidinones that might pos-
sess various biological activities.
to the literature, the hydrazones may exist as E/Z geometrical
–
isomers around C N double bonds and as cis/trans amide
–
conformers [32]. The 1H NMR spectra of compounds 3–15
displayed the resonance of a hydrazone N–H at 11.04–
11.55 ppm in the Z isomer and at 11.21–11.74 ppm in the
E isomer. Azomethine protons of the compounds resonated
at 7.92–8.30 ppm in the E isomer and at 8.15–8.46 ppm in the
Z isomer in dimethyl sulfoxide-d6 (DMSO-d6) solution. The
percentage of each isomer was calculated using the integral
values of the peak pairs and the dominating isomer was
assigned to the Z isomer due to azomethine protons. Also,
–CH3 protons at the C1 position were observed as two triplets
due to the canonic form. In addition, the NH protons of
compound 10 were observed to exchange with D2O in the
spectrum. All the other aliphatic and aromatic protons were
observed in the expected regions. The heteronuclear multiple
bond correlation (HMBC) spectrum of compound 9 also con-
firmed the detection of long-range 1H–13C couplings. The
structure of hydrazide-hydrazone 9 was established by using
an HMBC experiment as shown in Fig. 1.
Results
Chemistry
(R,S) 2-[1,8-Diethyl-1,3,4-tetrahydrapyrano(3,4-b)indole-1-
yl]acetic acid (etodolac), which possesses analgesic, antipy-
retic, and anti-inflammatory activities, was chosen as the
starting compound to design hydrazide-hydrazone and 4-
thiazolidinone derivatives (Scheme 1). Methyl(1,8-diethyl-
1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetate 1 was syn-
thesized by the reaction of etodolac and methanol in the
presence of a few drops of concentrated sulfuric acid. By
heating this methyl ester with hydrazine-hydrate in meth-
anol, we obtained 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-
b]indole-1-yl)acetohydrazide 2. After condensing hydrazide 2
with aromatic aldehydes in ethanol, 2-(1,8-diethyl-1,3,4,9-tet-
rahydropyrano[3,4-b]indole-1-yl)acetic acid[(5-nitro-2-furyl/
substituted phenyl)methylene]hydrazides 3–15 were
obtained in good yields (59–98%). 3-(2-(1,8-Diethyl-1,3,4,9-tet-
rahydropyrano[3,4-b]indol-1-yl)acetylhydrazono)-2-alkyl/aryl-
4-thiazolidinones 16–26 were obtained in moderate yields
(15–98%) by refluxing the related etodolac hydrazide-hydra-
zones 3–15 and thioglycolic acid in dry toluene for 9–16 h
4-Thiazolidinones 16–26 were prepared by cyclization of
the etodolac hydrazide-hydrazones 3–15 with thioglycolic
acid. The reaction proceeded by nucleophilic attack of the
anion of thioglycolic acid upon the carbon atom of a Schiff
base, followed by the capture of a proton by nitrogen and
cyclization of the latter with removal of a water molecule,
using a Dean–Stark water separator. Formation of 4-thiazo-
lidinones 16–26 was confirmed by IR spectroscopy, which
–
showed the ring C O stretching characteristic of lactam
–
in the range of 1705–1722 cmꢀ1 and amide C O bands at
–
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Etodolac Hydrazide Derivatives as Anticancer Agents
369
H3C
CH3
CH3
H
N
NH
CH3
O
O
H
N
CH3
HN
O
CH 3OH / d H2SO4
NH 2NH 2.H2
O
O
OH
O
C2H5OH
O
CH3
O
CH3
NH2
Etodolac
1
2
ArCHO/C2H5OH
CH3
COMPOUND
(Lab. Code)
Ar
H
N
CH3
O
_
2
(SGK 197)
O
HN
3
16
N
3−15
(SGK198) (SGK211)
NO 2
O
Ar-
OH
4
17
HS-CH 2-COOH / dry toluene
CH3
(SGK199) (SGK212)
CH3
H
N
5
18
F
(SGK200) (SGK213)
O
Ar-
F
F
O
HN
F
N
S
6
19
16−26
(SGK201) (SGK214)
O
7
20
(SGK203) (SGK216)
F
F
8
21
(SGK204) (SGK217)
F
9
22
(SGK205) (SGK218)
Cl
10
23
(SGK206) (SGK219)
OCH 3
F
11
24
(SGK207) (SGK220)
Cl
12
25
(SGK208) (SGK221)
NO 2
13
26
N
(SGK210) (SGK223)
14
(SGK209)
F
15
Scheme 1. Synthetic route of compounds 1–26.
(SGK202)
1653–1682 cmꢀ1 [33]. The structures of compounds 16–26
were supported by IR spectra as observed in 3–15 with the
Due to magnetic nonequivalance caused by the chiral center
produced by the nucleophilic addition of thioglycolic acid to
disappearance of 1601–1630 cmꢀ1 for the –N CH band and
the N CH function of etodolac hydrazide-hydrazones, the
–
–
–
–
1705–1722 cmꢀ1 for the C O of thiazolidinone [34–37].
methylene groups of etodolac 4-thiazolidinones appeared
as two doublets with coupling constants characteristic for
geminal protons (J ¼ 14.2–17.7 Hz). Additional support for
the structure of the 4-thiazolidinones was obtained from the
absorption positions of the N–CH–S protons, which showed
upfield shifts and resonated at about 5.13–6.41 ppm due to
the loss of the sp2 character of the involved C-atom [38–41].
The appearance of the C5–H and C2–H protons, characteristic
of thiazolidinones, confirmed the addition of thioglycolic
–
–
The 1H NMR and 13C NMR data were also in agreement with
the formation of the 4-thiazolidinone ring. The 1H NMR
–
spectra of compounds 16–26 lack the CH N signal at 7.92–
–
8.46 ppm, providing confirmatory evidence for the ring
closure from hydrazones 3–15. The 1H NMR spectra of the
4-thiazolidinones displayed resonances assigned to the
heterocyclic methylene ring protons at 3.66–3.94 and
3.82–4.12 ppm, corresponding to two diastereotopic protons.
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P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Figure 1. HMBC spectrum of compound 9.
acid across the azomethine linkage in the compounds. The
chemical shifts of the aromatic and NH protons of the com-
pounds have the expected values. The 13C NMR spectra of the
compounds 17, 18, 20, 21, 23, 25, and 26, which were chosen
as prototypes, verified the proposed 4-thiazolidinone struc-
ture [34, 41]. The peaks resonated at 30.95–37.07, 60.90–
65.92, 163.00–171.4, and 169.34–172.00 ppm for S–CH2,
guidelines.html). The anticancer screening data at a concen-
tration of 10ꢀ5 M are summarized in Fig. 2. In general, com-
pound 9 displayed antiproliferative activity against three
cancer cell lines (renal cancer, UO31; prostate cancer, PC-3;
non-small-cell lung cancer, HOP-92). The most potent derivative
was identified as 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-
b]indole-1-yl)acetic acid[(4-chlorophenyl)methylene]hydrazide
(compound 9), which demonstrated the most marked effect
on the prostate cancer cell line PC-3 (cell growth promotion
41.76%, inhibition 58.24%). However, compound 1, the start-
ing compound for synthesis, showed lower activity against
the PC-3 cell line. Moreover, compound 9 demostrated strong
cytotoxicity against the melanoma cell line SK-MEL-28.
The cell viability and growth inhibition (L-929 and PC-3)
was studied at the Marmara University Faculty of Pharmacy,
–
–
–
S–CH–N, amide C O, and thiazolidinone C O, respectively,
–
in the 13C NMR spectra of the 4-thiazolidinones. High-resol-
ution mass spectra (HR-MS) confirmed the molecular weights
and empirical formulae of the compounds and fragments,
with less than 5 mmu bias between the calculated and exper-
imental m/z values of either the molecular or the fragment
ions. The ionization mode was electron impact (EI) for all
4-thiazolidinone derivatives. The fragmentation pattern for
all etodolac 4-thiazolidinones (16–26) supported the pro-
posed structures. The fragment ion peaks, especially of
[M–CH2S]þ, [M–COCH2S]þ, and [(M þ 1)–S]þ, are the diagnos-
tic peaks for thiazolidinone rings [34, 42].
Biological activity
The primary anticancer assay was performed in accordance
with the protocol of the Drug Evaluation Branch, National
Cancer Institute (NCI), Bethesda [43–45]. As most of the com-
pounds in the submitted series of structures include one or
more functional groups that have been found troublesome in
the development of successful drug candidates, compounds
1, 9, 10, 20, and 21 were chosen as prototypes and evaluated
against the full panel of 60 human tumor cell lines at a single
dose for screening of their anticancer potential. In addition,
the selection criteria guidance is available online at the DTP
web site (www.dtp.nci.nih.gov/docs/misc/common_files/
Figure 2. Growth inhibition data of compounds 1, 9, 10, 20, and 21
at 10ꢀ5 M concentration.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Etodolac Hydrazide Derivatives as Anticancer Agents
371
involved in this process. This part of the study was performed
at the Department of Biochemistry, Faculty of Pharmacy,
Marmara University. Apoptosis is one of the major mechan-
isms of cell death in response to cancer therapies. Signaling
for apoptosis occurs through multiple independent path-
ways that are initiated by diverse extracellular and intracellu-
lar factors. The intrinsic cell death pathway is also known as
the mitochondrial apoptotic pathway [46]. It is known that
many apoptotic biochemical processes are regulated by bio-
chemical signaling agents found within mitochondria.
Mitochondria also play a central role in the integration
and propagation of death signals originating from inside
the cell, such as DNA damage and oxidative stress [47, 48].
In this study, the effects of etodolac and compound 9 on
apoptosis were investigated via determination of the depolar-
ization of the mitochondrial membrane potential and by
assessing the Bcl-2 and caspase-3 levels. As shown in Fig. 4,
after the cells were treated with etodolac and compound 9,
the depolarization of the mitochondrial membrane potential
increased as compared with the control group (p < 0.01) (a
colored version of Fig.
4 is provided as Supporting
Information Figure S1). At the same time, compound 9 sig-
nificantly increased the depolarization of the mitochondrial
membrane potential when compared with etodolac. Figure 4
shows the alterations of the mitochondrial membrane poten-
tial in PC-3 cells after treatment with etodolac and compound
9. The mitochondrial membrane potential changes were
determined using the J-aggregate-forming lipophilic cationic
fluorochrome 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzi-
midazole carbocyanine iodide (JC–1). The dye accumulates
and aggregates in mitochondria, emitting a bright red fluor-
escence. In apoptotic and necrotic cells with altered mito-
chondrial membrane potential, the dye remains in the
cytoplasm in its monomeric form and the fluorescence is
green. Loss of mitochondrial potential was indicated by
increased green fluorescence. Etodolac and compound 9
increased the loss of mitochondrial potential as compared
with the control group. The Bcl-2 levels of the etodolac and
compound 9 groups were significantly lower than those of
the control group (p < 0.01; Fig. 5). There were no significant
differences between etodolac and compound 9 in terms of the
Bcl-2 levels. The caspase-3 levels are shown in Fig. 6. Etodolac
and compound 9 caused a significant increase in the caspase-
3 activity as compared with the control group (p < 0.05 and
0.01, respectively). There were no significant differences
between etodolac and compound 9 in terms of the caspase-
3 levels.
Figure 3. (A) Effects of etodolac and compound 9 on the viability of
L-929 and PC-3 cells. (B) Effects of compound 9 on the viability of
PC-3 cancer cells.
Department of Pharmaceutical Biotechnology. On the basis
of the above results, the cell viability assessment was per-
formed by measuring the levels of live cells after incubation
with different doses of etodolac and compound 9 for 48 h,
using the MTT colorimetric assay against a panel of L-929 rat
fibroblast cell (ATCC, CCL-1) and PC-3 prostate cancer cell
(ATCC, CRL-1435) lines (100 indicates no activity; 0 indicates
complete cell death) at a final DMSO concentration of 0.1–
0.05%. The cell viability results are presented in Fig. 3.
When compared to etodolac, which was used as standard,
compound 9 did not cause a significant effect on the viability
of L-929 rat fibroblasts at the applied doses. This clearly
indicates that the hydrazide-hydrazone structure of com-
pound 9 does not have higher toxic effects toward L-929
rat fibroblasts. Compound 9 also gave rise to a significant
change in cell viability in the PC-3 prostate cancer cell line at
different doses, compared to the standard drug etodolac.
Compound 9, having a hydrazide-hydrazone moiety, was
responsible for a significant decrease in cell viability com-
pared to etodolac. The growth inhibitory property (IC50) for
compound 9 was 54 mM (22.842 mg/mL).
Loss of mitochondrial membrane potential and release of
mitochondrial proteins are of central importance in media-
ting and enhancing apoptotic pathways. In this study, we
investigated the effects of etodolac and compound 9 on the
mitochondrial membrane potential and Bcl-2 as mitochon-
In the present study, we aimed at investigating whether
the influence of compound 9 and etodolac occurred via the
apoptotic pathway and, if so, which apoptotic pathway was
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372
P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Figure 4. (A) Effects of etodolac and com-
pound 9 on the mitochondrial membrane poten-
tial in PC-3 cells
(
ꢁꢁp < 0.01 vs. control,
þp < 0.05 vs. etodolac). (B) Mean values of
JC-1 green fluorescence for etodolac and com-
pound 9, indicating the mitochondrial mem-
brane potential in the PC-3 cell line.
(Fluorescence microscopy after JC-1 staining
showing changes in the mitochondrial mem-
brane potential in PC-3 cells; magnification:
ꢂ200.)
drial apoptotic markers and on caspase-3 activation as
marker for the point of convergence of the intrinsic and
extrinsic apoptotic pathways in the PC-3 cell line. Our data
show that 24-h treatment with etodolac and compound 9
directly down-regulated Bcl-2 expression and induced
caspase-3-dependent apoptosis in the PC-3 cell line.
Additionally, these agents caused mitochondrial membrane
potential loss in the PC-3 cells. It has been shown that eto-
dolac induced a disturbance of the mitochondrial membrane
potential and caused caspase-9, -7, and -3 activation in
addition to the reduction of Bcl-2 levels in myeloma cells
[49]. The central function of mammalian Bcl-2 family mem-
bers is to guard the mitochondrial integrity and to control
the release of mitochondrial proteins into the cytoplasm [50].
Bcl-2, an anti-apoptotic protein, prevents the release of cyto-
chrome c from mitochondria and the activation of caspases,
resulting in anti-apoptotic action and the maintenance of
cell survival [51]. Our findings indicate that etodolac and
Figure 5. Effects of etodolac and compound 9 on Bcl-2 in PC-3
cells (ꢁꢁp < 0.01 vs. control).
Figure 6. Effects of etodolac and compound 9 on caspase-3 activ-
ity in PC-3 cells (ꢁp < 0.05 vs. control, ꢁꢁp < 0.01 vs. control).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Etodolac Hydrazide Derivatives as Anticancer Agents
373
compound 9 induce apoptosis in human PC-3 prostate cancer
Methyl (1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-
1-yl)acetate (1)
cells through the mitochondrial pathway.
Etodolac (0.01 mol) and methanol (16 mL) were refluxed for 3 h
in a few drops of concentrated sulfuric acid. The contents of the
flask were subsequently cooled and neutralized by using NaHCO3
(5%). The resulting precipitate was filtered, dried, and recrystal-
lized twice from ethanol. Cream-colored solid. MW: 301.380. m.p.
Discussion
In this study, etodolac hydrazide and a series of novel eto-
dolac hydrazide derivatives were synthesized and evaluated
for their anticancer activity. After a preliminary survey, com-
pound 9 was selected by the NCI for anticancer screening.
This compound was found to show significant inhibition on
the prostate cancer cell line PC-3.
128–1308C [28, 29]. Yield 66%. Rf ꢂ 100 value 87 (M1). IR (vmax
,
1
cmꢀ1): 3379 (indole NH), 1705 (C O). H NMR (400 MHz, DMSO-
–
–
d6): d (ppm) 0.83 (3H, t, –CH2–CH3 at C1); 1.37 (3H, t, –CH2–CH3
at C8); 1.98–2.18 (2H, m, –CH2–CH3 at C1); 2.73–3.04 (6H, m,
–CH2–CH3 at C8, –CH2–COOCH3 at C1 and –CH2 at C4); 3.72
(3H, s, –COOCH3); 3.94–4.06 (2H, m, –CH2 at C3); 7.01 (1H,
d, C7–H, J ¼ 6.8 Hz); 7.06 (1H, t, C6–H); 7.36 (1H, d, C5–H,
J ¼ 7.2 Hz); 9.06 (s, 1H, indole N–H). Anal. calcd.
for C18H23NO3: C, 71.73; H, 7.69; N, 4.65%. Found: C, 70.85; H,
7.24; N, 4.75%.
In this study, we identified compound 9 as a promising
lead compound for anticancer drug development. This study
indicates that the synthesis of hydrazone and 4-thiazolidi-
none derivatives of etodolac resulted in more effective com-
pounds compared to the starting compound, etodolac. Based
on these results, we are now in the process of synthesizing
modified analogs of the lead compound in order to generate
more effective anticancer agents. The results from the anti-
cancer and apoptosis studies on compound 9, which was
synthesized from racemic etodolac, indicate the need for
an evaluation of the above-mentioned activities of the S- and
R-isomers of hydrazones. Thus, the apoptosis results obtained
from this planned study will provide an explanation of the
apoptotic pathway based on enantiomeric products.
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetohydrazide (2)
To a methanolic solution of compound 1 (20 mL, 0.01 mol) was
added hydrazine-hydrate (80%, 7 mL) and refluxed for 3 h. The
reaction mixture was then cooled, diluted with water and
allowed to stand overnight. The precipitated solid was washed
with water, dried, and recrystallized twice from petroleum
ether.
White colored solid. MW: 301.383. m.p. 186–1888C. Yield 80%.
Rf ꢂ 100 value 51 (M1). FT-IR (vmax, cmꢀ1): 3354, 3310 (indole and
1
hydrazide NH), 1651 (C O). H NMR (400 MHz, DMSO-d ): d (ppm)
–
–
6
0.62 (3H, t, –CH2–CH3 at C1); 1.26 (3H, t, –CH2–CH3 at C8); 2.00–
2.07 (2H, m, –CH2–CH3 at C1); 2.58–2.86 (6H, m, CH2–CONHNH2,
–CH2–CH3 at C8 and –CH2 at C4); 3.91–3.97 (2H, m, –CH2 at C3);
4.27 (2H, b.s., NH–NH2); 6.87–7.24 (3H, m, Ar–H); 8.94
(1H, s, NHNH2); 10.55 (s, 1H, indole N–H). Anal. calcd.
for C17H23N3O2: C, 67.75; H, 7.69; N, 13.94%. Found: C, 67.33;
H, 6.95; N, 13.73%.
Experimental
Chemistry
All chemicals were purchased from Merck, Sigma–Aldrich, or
Fluka. Reactions were monitored by TLC on silica gel plates
purchased from Merck. Melting points of the synthesized com-
pounds were determined in a Schmelzpunktgera¨t SMP II melting
point apparatus and are uncorrected. The purity of the com-
pounds was checked on TLC plates precoated with silica gel G
using the solvent systems M1 (petroleum ether/acetone 60:40 v/v),
M2 (petroleum ether/ethyl acetate 60:40 v/v), M3 (petroleum
ether/ethyl acetate 50:50 v/v), M4 (petroleum ether/ethyl acetate
70:30 v/v), M5 (petroleum ether/ethyl acetate 30:70 v/v), M6
(chloroform/methanol 80:20 v/v), and M7 (chloroform/methanol
60:40 v/v). The spots were located under UV light (254 nm)
General procedure for the synthesis of 2-(1,8-diethyl-
1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetic acid[(5-
nitro-2-furyl/substituted phenyl)methylene]hydrazides
3–15
A solution of compound 2 (0.01 mol) and equimolar amounts of
appropriate aromatic aldehyde in absolute ethanol (30 mL) was
heated under reflux for 3 h. The obtained precipitate was filtered
off, dried, and recrystallized twice from ethanol.
(T ¼ 218C). Elemental analyses were performed on
a
VarioMICRO V1.5.7. instrument. FT-IR spectra were recorded
1
on a Shimadzu FT-IR-8400S spectrophotometer. H NMR spectra
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(5-nitro-2-furyl)methylene]hydrazide (3)
Dark orange solid. MW: 425.450. m.p. 165–1678C. Yield 96%.
Rf ꢂ 100 value: 22 (M2). FT-IR (vmax, cmꢀ1): 3449, 3349, 3214
were recorded on Bruker Avance-DPX 400 (400 MHz), Bruker
Avance 500 (500 MHz), and Varian NMR Mercury 300
(300 MHz) NMR spectrometers using DMSO-d6 as solvent.
MALDI-TOF HR-MS spectra using the EI and FAB ionization tech-
niques were performed using a Jeol JMS-700 instrument. Mass
spectra (MS) were determined on an Agilent 1100 LC-MS mass
spectrometer. Chemical shifts (d) are reported in parts per
million (ppm). Data are reported as follows: chemical shift,
multiplicity (b.s.: broad singlet, d: doublet, m: multiplet, s: sin-
glet, and t: triplet), coupling constants (Hz), integration. 13C NMR
spectra were recorded on Bruker Avance-DPX 100 (100 MHz) and
Bruker Avance-75 (75 MHz) spectrometers.
(indole and hydrazone NH), 1653 (C O), 1607 (C N). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.58, 0.67 (3H, tt, –CH2–CH3 at C1);
1.18–1.28 (3H, m, –CH2–CH3 at C8); 2.04–2.13 (2H, m, –CH2–CH3
at C1); 2.61–2.69 (2H, m, –CH2–CH3 at C8); 2.83–3.72 (m, 4H,
–CH2–CONH and –CH2 at C4); 3.81–4.09 (2H, m, –CH2 at C3);
–
6.87–7.78 (5H, m, Ar–H); 7.92, 8.24 (1H, ss, –N CH); 10.51 (1H,
–
d, indole N–H); 11.55, 11.74 (1H, ss, –CO–NH). Anal. calcd.
for C22H24N4O5: C, 62.25; H, 5.70; N, 13.20%. Found: C, 62.12;
H, 5.71; N, 12.95%.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

374
P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.24–1.29 (3H, m,
–CH2–CH3 at C8); 2.06–2.19 (2H, m, –CH2–CH3 at C1); 2.56–2.70
(2H, m, –CH2–CH3 at C8); 2.81–3.71 (4H, m, –CH2CONH at C1 and
–CH2 at C4); 3.82–4.03 (2H, m, –CH2 at C3); 6.87–7.52 (6H, m,
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(2-hydroxy phenyl)methylene]hydrazide (4)
Light cream-colored solid. MW: 405.490. m.p. 1608C. Yield 79%.
Rf ꢂ 100 value: 29.51 (M2). FT-IR (vmax, cmꢀ1): 3379, 3264 (indole
–
Ar–H); 8.13, 8.38 (1H, ss, –N CH); 10.47 (1H, d, indole N–H); 11.33,
–
11.47 (1H, ss, –CO–NH). Anal. calcd. for C24H25F2N3O2: C, 67.75; H,
and hydrazone NH, OH), 1667 (C O), 1618 (C N). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.60–0.68 (3H, m, –CH2–CH3
at C1); 1.24–1.29 (3H, m, –CH2–CH3 at C8); 2.07–2.12 (2H, m,
–CH2–CH3 at C1); 2.62–2.70 (2H, m, –CH2–CH3 at C8); 2.82–2.96
(4H, m, –CH2CONH and –CH2 at C4); 3.54–4.01 (2H, m, –CH2 at C3);
5.92; N, 9.88%. Found: C, 67.67; H, 5.90; N, 9.69%.
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
–
6.88–7.58 (7H, m, Ar–H); 8.30, 8.42 (1H, ss, N CH); 10.08, 10.51
–
yl)acetic acid[(4-chlorophenyl)methylene]hydrazide (9)
White solid. MW: 423.935. m.p. 1748C. Yield 80%. Rf ꢂ 100 value:
36.07 (M2). FT-IR (vmax, cmꢀ1): 3231, 3213 (indole and hydrazone
(1H, ss, indole N–H); 11.18, 11.29 (1H, s, CO–NH); 11.42 (1H, s,
–OH). Anal. calcd. for C24H27N3O3: C, 71.09; H, 6.71; N, 10.36%.
Found: C, 70.98; H, 6.76; N, 10.21%.
NH), 1659 (C O), 1612 (C N). 1H NMR (400 MHz, DMSO-d6):
–
–
–
–
d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.24–1.28 (3H, m,
–CH2–CH3 at C8); 2.07–2.15 (2H, m, –CH2–CH3 at C1); 2.60–2.69
(2H, m, –CH2–CH3 at C8); 2.81–2.98 (m, 4H, –CH2CONH at C1 and
–CH2 at C4); 3.70–4.01 (2H, m, –CH2 at C3); 6.87–7.71 (7H, m,
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)-
acetic acid[(4-trifluoromethylphenyl)methylene]-hydrazide (5)
Cream-colored solid. MW: 466.496. m.p. 1408C. Yield 86%.
Rf ꢂ 100 value: 41.93 (M2). FT-IR (vmax, cmꢀ1): 3424, 3358,
–
Ar–H); 7.98, 8.22 (1H, ss, –N CH–); 10.51 (1H, d, indole N–H);
–
11.23, 11.39 (1H, ss, –CO–NH). Anal. calcd. for C24H26ClN3O2: C,
–
–
–
3312 (indole and hydrazone NH), 1651 (C O), 1622 (C N).
–
68.00; H, 6.18; N, 9.91%. Found: C, 68.14; H, 6.27; N, 9.89%.
1H NMR (400 MHz, DMSO-d6): d (ppm) 0.60, 0.67 (3H, tt,
–CH2–CH3 at C1); 1.26 (3H, t, –CH2–CH3 at C8); 2.02–2.10
(2H, m, –CH2–CH3 at C1); 2.54–2.66 (2H, m, –CH2–CH3 at C8);
2.83–3.01 (4H, m, –CH2CONH– at C1 and –CH2 at C4); 3.56–3.92
(2H, m, –CH2 at C3); 6.89–7.90 (7H, m, Ar–H); 8.06, 8.30 (1H, ss,
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(4-methoxyphenyl)methylene]acetohydrazide
(10)
–
–N CH); 10.51 (d, 1H, indole N–H); 11.36, 11.53 (1H, ss,
–
–CO–NH–). Anal. calcd. for C25H26F3N3O2.1/2H2O: C, 64.37; H,
White solid. MW: 419.516. m.p. 171–1738C. Yield 76%. Rf ꢂ 100
value: 44.44 (M3). FT-IR (vmax, cmꢀ1): 3297, 3260 (indole and
5.83; N, 9.01%. Found: C, 64.87; H, 5.19; N, 8.37%.
hydrazone NH), 1647 (C O), 1605 (C N). 1H NMR (400 MHz,
–
–
–
–
DMSO-d6): d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.06–1.29
(3H, m, –CH2–CH3 at C8); 2.07–2.16 (2H, m, –CH2–CH3 at C1); 2.60–
2.70 (m, 2H, CH2–CH3 at C8); 2.78–3.71 (m, 4H, –CH2CONH at C1
and –CH2 at C4); 3.80 (3H, s, –OCH3); 3.84–4.01 (2H, m, –CH2 at C3);
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(2-fluorophenyl)methylene]hydrazide (6)
Light cream-colored solid. MW: 407.480. m.p. 1388C. Yield 65%.
Rf ꢂ 100 value: 50.82 (M2). FT-IR (vmax, cmꢀ1): 3302 (indole and
–
6.85–7.63 (7H, m, Ar–H); 7.94, 8.15 (1H, ss, N CH); 10.51 (1H, d,
–
hydrazone NH), 1645 (C O), 1605 (C N). 1H NMR (400 MHz,
indole N–H); 11.04, 11.21 (1H, ss, –CO–NH). Anal. calcd. for
C25H29N3O3: C, 71.57; H, 6.97; N, 10.02%. Found: C, 71.35; H,
6.84; N, 9. 91%. MS [API-ES, m/z (%)]: 420.3 ([Mþ]þ1, 4.2), 419.3
(25.7), 418.2 (100), 225.5 (8.8), 169.3 (2.2).
–
–
–
–
DMSO-d6): d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.24–1.28
(3H, m, –CH2–CH3 at C8); 2.08–2.14 (2H, m, –CH2–CH3 at C1); 2.60–
2.69 (2H, m, –CH2–CH3 at C8); 2.81–3.55 (4H, m, –CH2CONH at C1
and –CH2 at C4); 3.69–4.01 (2H, m, –CH2 at C3); 6.87–7.89 (7H, m,
–
Ar–H); 8.21, 8.46 (1H, ss, –N CH); 10.51 (1H, s, indole N–H); 11.33,
–
11.46 (1H, ss, –CO–NH). Anal. calcd. for C24H26FN3O2: C, 70.74; H,
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(2-chloro-6-fluorophenyl)methylene]hydrazide
(11)
6.43; N, 10.31%. Found: C, 70.35; H, 5.84; N, 10.17%.
Cream-colored solid. MW: 441.926. m.p. 151–1538C. Yield 59%.
Rf ꢂ 100 value: 41.94 (M4). FT-IR (vmax, cmꢀ1): 3324, 3232 (indole
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(4-fluorophenyl)methylene]hydrazide (7)
Cream-colored solid. MW: 407.480. m.p. 1488C. Yield 98%.
1
–
–
–
and hydrazone NH), 1649 (C O), 1603 (C N). H NMR (400 MHz,
–
DMSO-d6): d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.26 (3H, t,
–CH2–CH3 at C8); 2.08–2.17 (2H, m, –CH2–CH3 at C1); 2.56–2.68
(2H, m, –CH2–CH3 at C8); 2.82–3.68 (4H, m, –CH2CONH at C1 and
–CH2 at C4); 3.84–4.02 (2H, m, –CH2 at C3); 6.87–7.47 (6H, m,
–
Rf ꢂ 100 value: 31.15 (M2). FT-IR (vmax, cmꢀ1): 3379, 3239 (indole
1
–
–
–
and hydrazone NH), 1661 (C O), 1601 (C N). H NMR (400 MHz,
–
DMSO-d6): d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.24–1.28
(3H, m, –CH2–CH3 at C8); 2.05–2.15 (2H, m, –CH2–CH3 at C1); 2.60–
2.69 (2H, m, –CH2–CH3 at C8); 2.80–3.56 (4H, m, –CH2CONH at C1
and –CH2 at C4); 3.69–4.01 (2H, m, –CH2 at C3); 6.87–7.75 (7H, m,
Ar–H); 8.25, 8.46 (1H, ss, –N CH); 10.47 (d, 1H, indole N–H); 11.41,
–
11.54 (1H, ss, –CO–NH). Anal. calcd. for C24H25ClFN3O2: C, 65.23;
H, 5.70; N, 9.51%. Found: C, 65.50; H, 5.56; N, 9.31%.
–
Ar–H); 7.99, 8.22 (1H, ss, –N CH–); 10.51 (1H, d, indole N–H);
–
11.17, 11.34 (1H, ss, –CO–NH). Anal. calcd. for C24H26FN3O2: C,
70.74; H, 6.43; N, 10.31%. Found: C, 70.58; H, 6.39; N, 9.92%.
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(4-nitrophenyl)methylene]hydrazide (12)
Orange solid. MW: 434.488. m.p. 2468C. Yield 75%. Rf ꢂ 100
value: 21.31 (M2). FT-IR (vmax, cmꢀ1): 3418 (indole and hydrazone
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
NH), 1661 (C O), 1611 (C N). 1H NMR (400 MHz, DMSO-d6):
yl)acetic acid[(2,6-difluorophenyl)methylene]hydrazide (8)
Light brown solid. MW: 425.471. m.p. 1458C. Yield 94%. Rf ꢂ 100
value: 47.54 (M2). FT-IR (vmax, cmꢀ1): 3323 (indole and hydrazone
–
–
–
–
d (ppm) 0.59, 0.67 (3H, tt, –CH2–CH3 at C1); 1.24–1.28 (3H, m,
–CH2–CH3 at C8); 2.08–2.14 (2H, m, CH2–CH3 at C1); 2.61–2.68
(2H, m, –CH2–CH3 at C8); 2.83–3.76 (m, 4H, –CH2CONH at C1 and
NH), 1645 (C O), 1624 (C N). 1H NMR (400 MHz, DMSO-d6):
–
–
–
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Etodolac Hydrazide Derivatives as Anticancer Agents
375
–
amide NH), 1722 (thiazolidinone, C O), 1668 (amide, C O).
–
–
–CH2 at C4); 3.78–4.01 (2H, m, –CH2 at C3); 6.87–8.30 (7H, m,
Ar–H); 8.08, 8.34 (1H, ss, N CH); 10.51 (1H, d, indole N–H); 11.46,
–
–
1H NMR (300 MHz, DMSO-d6):
d (ppm) 0.54–1.24 (m, 6H,
–
11.57 (1H, ss, CO–NH). Anal. calcd. for C24H26N4O4: C, 66.34; H,
6.03; N, 12.89%. Found: C, 66.10; H, 5.19; N, 12.83%.
–CH2–CH3 at C1 and C8); 1.89–3.00 (m, 10H, –CH2–CH3 at C1
and C8; –CH2–CO–NH at C1; –CH2 at C3 and C4); 3.76, 3.89 (2d
and each 1H, –S–CH2–, J ¼ 15.6 and 15.9 Hz); 5.15–5.92 (m, 1H,
N–CH–S); 6.83–7.67 (m, 5H, Ar–H); 10.13–10.40 (m, 2H, indole
NH and –CO–NH–N–). HR-MS (EIþ) Calcd./Found (m/z): 498.1573/
498.1610 (Mþ) (C24H26N4O6S); 469.1181/469.1192 (C22H21N4O6S);
467.1930/467.1423; 286.1681/286.1608; 270.1488/270.1434;
242.1539/242.1531; 242.1171/242.1171; 240.1019/240.1048
(C15H14NO2); 229.0157/229.1405; 229.1466/229.1433; 228.1382/
228.1382 (C15H18NO); 227.1310/227.1322; 212.1069/212.1246.
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(pyridine-4-yl)methylene]hydrazide (13)
Yellow solid. MW: 408.493. m.p. 112–1138C. Yield 88%. Rf ꢂ 100
value: 15.87 (M3). FT-IR (vmax, cmꢀ1): 3424, 3308 (indole and
hydrazone NH), 1653 (C O), 1630 (C N). 1H NMR (400 MHz,
–
–
–
–
DMSO-d6): d (ppm) 0.57–0.69 (3H, m, –CH2–CH3 at C1); 1.24–1.28
(3H, m, –CH2–CH3 at C8); 2.07–2.13 (2H, m, –CH2–CH3 at C1); 2.61–
2.68 (2H, m, –CH2–CH3 at C8); 2.83–3.56 (4H, m, –CH2CONH at C1
and –CH2 at C4); 3.69–4.01 (2H, m, –CH2 at C3); 6.87–8.33 (m, 7H,
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)-acetylhydrazono)-2-(2-hydroxyphenyl)-4-thiazolidinone
(17)
–
Ar–H); 7.96, 8.23 (1H, ss, N CH); 10.50 (1H, d, indole N–H); 11.42,
–
11.58 (1H, ss, –CO–NH). Anal. calcd. for C23H26N4O4 H2O: C, 67.63;
ꢃ
H, 6.91; N, 13.72%. Found: C, 67.34; H, 6.60; N, 12.87%.
Orange solid. MW: 479.591. m.p. 105–1078C. Yield 65%. Rf ꢂ 100
value: 8 (M5). FT-IR (vmax, cmꢀ1): 3254 (indole and amide NH, OH),
1717 (thiazolidinone, C O), 1653 (amide, C O). 1H NMR
–
–
–
–
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid(phenyl methylene)hydrazide (14)
(500 MHz, DMSO-d6): d (ppm) 0.49–1.25 (m, 10H, –CH2–CH3
at C1 and C8); 2.03–3.13 (m, 6H, –CH2–CO–NH at C1, –CH2
at C3 and C4); 3.66, 4.08 (2d and each 1H, –S–CH2–, J ¼ 16.0
and 16.5 Hz); 5.59–6.06 (m, 1H, –N–CH–S); 6.83–7.39 (m, 7H,
Ar–H); 9.89–10.39 (m, 3H, indole NH, –CO–NH–N and –OH).
13C NMR (100 MHz) (DMSO-d6/TMS): d (ppm) 8.34 (C-12); 14.57
(C-10); 15.01 (C-9); 22.41 (C-4); 23.76 (C-11); 31.25 (thiazolidinone
S–CH2–); 57.37 (C-13); 60.88 (C-3); 65.92 (thiazolidinone –S–CH–);
76.72 (C-1); 108.31 (C-4a); 116.57 (C-10); 118.72 (C-6); 119.92 (C-5);
120.86 (C-30); 127.27 (C-60); 127.84 (C-7); 130.25 (C-5a); 130.57 (C-50);
135.73 (C-40); 137.53 (C-8); 139.27 (C-1a); 142.82 (C-8a); 168.50 (Cþ-
Light cream-colored solid. MW: 398.498. m.p. 127–1298C. Yield
68%. Rf ꢂ 100 value: 34.43 (M2). FT-IR (vmax, cmꢀ1): 3304 (indole
1
–
–
–
and hydrazone NH), 1639 (C O), 1605 (C N). H NMR (400 MHz,
–
DMSO-d6): d (ppm) 0.59, 0.67 (3H, tt, –CH2-CH3 at C1); 1.24–1.29
(3H, m, –CH2–CH3 at C8); 2.08–2.16 (2H, m, –CH2–CH3 at C1); 2.60–
2.68 (2H, m, –CH2–CH3 at C8); 2.80–2.99 (4H, m, –CH2CONH at C1
and –CH2 at C4); 3.71–4.01 (2H, m, –CH2 at C3); 6.88–7.69 (8H, m,
–
Ar–H); 8.00, 8.22 (1H, ss, –N CH); 10.51 (1H, d, indole N–H); 11.16,
–
11.34 (1H, ss, CO–NH). Anal. calcd. for C24H27N3O2 1/2 H2O: C,
ꢃ
72.33; H, 7.08; N, 10.54%. Found: C, 72.22; H, 6.60; N, 10.32%.
0
–
2 ); 171.14 (CO–NH); 172.00 (thiazolidinone C O). HR-MS (EI )
–
Calcd./Found (m/z): 479.1873/479.1909 (Mþ) (C26H29N3O4S);
405.2052/405.2036; 270.1488/270.1448; 242.1539/242.1153;
242.1175/242.1153; 240.1019/240.1068 (C15H14NO2); 229.1466/
229.1404; 228.1382/228.1366 (C15H18NO); 227.1310/227.1304;
212.1069/212.1248; 210.0463/210.1280.
2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetic acid[(3-fluorophenyl)methylene]hydrazide (15)
Light yellow solid. MW: 407.480. m.p. 156–1578C. Yield 86%.
Rf ꢂ 100 value: 36 (M2). FT-IR (vmax, cmꢀ1): 3279 (indole and
hydrazone NH), 1667 (C O), 1618 (C N). 1H NMR (400 MHz,
–
–
–
–
DMSO-d6): d (ppm) 0.59, 0.66 (3H, tt, –CH2–CH3 at C1); 1.24–1.28
(3H, m, –CH2–CH3 at C8); 2.07–2.13 (2H, m, –CH2–CH3 at C1); 2.60–
2.69 (2H, m, –CH2–CH3 at C8); 2.81–3.70 (4H, m, –CH2CONH at C1
and –CH2 at C4); 3.79–4.01 (2H, m, –CH2 at C3); 6.87–7.51 (7H, m,
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-trifluoromethylphenyl)-4-
thiazolidinone (18)
Yellow solid. MW: 531.590. m.p. 99–1018C. Yield 51%. Rf ꢂ 100
–
Ar–H); 7.98, 8.23 (1H, ss, –N CH); 10.51 (1H, s, indole N–H); 11.28,
–
11.43 (1H, ss, –CO–NH). Anal. calcd. for C24H26FN3O2 (407.480): C,
value: 40.62 (M5). FT-IR (vmax, cmꢀ1): 3290 (indole and amide NH),
1713 (thiazolidinone, C O), 1674 (amide, C O). 1H NMR
–
–
–
–
70.74; H, 6.43; N, 10.31%. Found: C, 70.15; H, 6.41; N, 9.92%.
(300 MHz, DMSO-d6): d (ppm) 0.50 (t, 3H, –CH2–CH3 at C1);
1.04–2.76 (m, 13H, –CH2–CH3 at C8; –CH2–CH3 at C1 and C8;
–CH2–CO–NH at C1; –CH2 at C3 and C4); 3.71, 3.93 (2d and each
1H, –S–CH2–, J ¼ 15.9 Hz); 5.13–5.88 (m, 1H, N–CH–S); 6.84–7.75
(m, 7H, Ar–H); 9.96, 10.06 (ss, 1H, indole NH); 10.31, 10.37
(ss, 1H –CO–NH–N). 13C NMR (75 MHz) (DMSO-d6/TMS): d (ppm)
8.11 (C-12); 14.90 (C-10); 22.23 (C-9); 24.16 (C-4); 39.70 (C-11);
31.32 (thiazolidinone S–CH2–); 42.49 (C-13); 60.51 (C-3); 61.44
(thiazolidinone –S–CH–); 75.69 (C-1); 107.71 (C-4a); 115.84 (C-6);
119.19 (C-5); 120.13 (C-7); 124.46 (–CF3); 125.90 (C-8); 126.42 (C-30
and C-50); 126.84 (C-5a); 128.92 (C-20 and C-60); 129.95 (C-40); 134.93
General procedure for the synthesis of 3-(2-(1,8-diethyl-
1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetylhydrazono)-
2-alkyl/aryl-4-thiazolidinones 16–26
A mixture of compounds 3–15 (0.01 mol) and thioglycolic acid
(0.2 mol) was refluxed in dry toluene (100 mL) by using a Dean–
Stark water separator. Excess toluene was evaporated under
vacuum and the flask content was neutralized by the addition
of NaHCO3 (5%) until the CO2 release was completed. The pre-
cipitated solid was washed with water, dried and recrystallized
from ethanol/water.
(C-1a); 136.51 (C-8a); 143.84 (C-10); 168.24 (CO–NH); 169.34
þ
–
(thiazolidinone C O). HR-MS (EI ) Calcd./Found (m/z): 531.1803/
–
531.1799 (Mþ) (C27H28F3N3O3S); 512.1814/512.1729; 502.1406/
502.1413 (C25H23F3N3O3S); 485.1926/485.1299; 270.1488/
270.1409; 262.0388/262.9926; 242.1539/242.1199; 242.1175/
242.1171; 240.1019/240.1063; 229.1466/229.1443; 228.1382/
228.1412 (C15H18NO); 227.1310/227.1313; 212.1069/212.1061.
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono-2-(5-nitro-2-furyl)-4-thiazolidinone (16)
Dark yellow solid. MW: 498.551. m.p. 101–1038C. Yield 29%.
Rf ꢂ 100 value: 71.43 (M8). FT-IR (vmax, cmꢀ1): 3325 (indole and
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

376
P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
0
–
and C-6 ); 163.00 (CO–NH); 169.53 (thiazolidinone C O). HR-MS
–
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(2-fluorophenyl)-4-thiazolidinone
(19)
(EIþ) Calcd./Found (m/z): 499.1741/499.1763 (Mþ) (C26H27F2N3O3S);
481.1835/481.1619; 468.2098/468.1543; 463.1930/463.2004;
453.1863/453.1369; 286.1681/286.1460; 270.1488/270.1431;
242.1539/242.1502; 242.1175/242.1139; 240.1019/240.1062
(C15H14NO2); 230.1488/230.1431; 229.1466/229.1398; 228.1382/
228.1373 (C15H18NO); 227.1310/227.1297; 212.1069/212.1423.
Orange solid. MW: 481.582. m.p. 106–1088C. Yield 98%. Rf ꢂ 100
value: 31.37 (M2). FT-IR (vmax, cmꢀ1): 3265 (indole and amide NH),
1
1720 (thiazolidinone, C O), 1674 (amide, C O). H NMR (300 MHz,
–
–
–
–
DMSO-d6): d (ppm) 0.45–1.22 (m, 6H, –CH2–CH3 at C1 and at C8);
1.84–3.11 (m, 10H, –CH2–CH3 at C1 and at C8; –CH2–CO–NH at C1,
–CH2 at C3 and C4); 3.82, 4.10 (2d and each 1H, –S–CH2–,
J ¼ 15.9 Hz,); 5.14–6.02 (m, 1H, N–CH–S); 6.86–7.52 (m, 7H,
Ar–H); 10.07–10.50 (m, 2H, indole NH and –CO–NH–N). HR-MS
(EIþ) Calcd./Found (m/z): 481.1835/481.1849 (Mþ) (C26H28FN3O3S);
463.1929/463.1688; 450.2187/450.1672; 435.1958/435.9727;
270.1488/270.1564; 242.1539/242.1474; 242.1175/242.1168;
240.1019/240.1076; 229.1466/229.1426; 228.1382/228.1374
(C15H18NO); 227.1310/227.1301; 212.1069/212.1067.
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-chlorophenyl)-4-thiazolidinone
(22)
Dark orange solid. MW: 498.037. m.p. 91–938C. Yield 44%.
Rf ꢂ 100 value: 31.25 (M5). FT-IR (vmax, cmꢀ1): 3287 (indole and
–
–
–
amide NH), 1705 (thiazolidinone, C O), 1674 (amide, C O).
–
1H NMR (400 MHz, DMSO-d6): d (ppm) 0.52–3.85 (m, 16H,
–CH2–CH3 at C1 and C8; –CH2–CO–NH at C1; –CH2 at C3
and C4); 3.87, 4.11 (2d and each 1H, –S–CH2–, J ¼ 16.3 and
16.8 Hz); 5.14–5.79 (m, 1H, N–CH–S); 6.62–7.84 (m, 7H, Ar–H);
9.94–10.41 (m, 2H, indole NH and –CO–NH–N–). HR-MS (EIþ)
Calcd./Found (m/z): 497.1539/497.1487 (Mþ) (C26H28ClN3O3S);
468.1148/468.1201 (C24H23ClN3O3S); 466.1891/466.1342;
463.1929/463.1892; 451.1662/451.1429; 286.1681/286.1356;
270.1488/270.1456; 242.1539/242.1577; 242.1175/242.1169;
240.1019/240.1075 (C15H14NO2); 229.1466/229.1393; 228.1382/
228.1360 (C15H18NO); 227.1310/227.1277; 212.1069/212.1037;
194.1681/194.0975.
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-fluorophenyl)-4-thiazolidinone
(20)
Orange solid. MW: 481.582. m.p. 133–1358C. Yield 75%. Rf ꢂ 100
value: 28.30 (M5). FT-IR (vmax, cmꢀ1): 3298 (indole and amide NH),
1713 (thiazolidinone, C O), 1674 (amide, C O). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.51–1.26 (m, 6H, –CH2–CH3 at C1
and C8); 1.6–3.71 (m, 10H, –CH2–CH3 at C1 and C8; –CH2–CO–NH
at C1; –CH2 at C3 and C4); 3.80, 3.84 (2d and each 1H, –S–CH2–,
J ¼ 16.02 Hz); 5.71, 5.81 (ss, 1H, N–CH–S); 6.86–7.50 (m, 7H,
Ar–H); 9.92, 10.01 (ss, 1H, indole NH); 10.32,10.38 (ss, 1H,
–CO–NH–N). 13C NMR (100 MHz) (DMSO-d6/TMS): d (ppm) 8.19
(C-12); 14.97 (C-10); 22.39 (C-9); 24.30 (C-4); 29.91 (C-11); 31.57
(thiazolidinone S–CH2–); 42.73 (C-13); 60.88 (C-3); 61.90 (thiazo-
lidinone –S–CH–); 76.15 (C-1); 108.29 (C-4a); 116.37 (C-6); 116.54
(C-5); 119.83 (C-30 ve C-50); 120.76 (C-7); 127.23 (C-5a); 127.69 (C-20
ve C-60); 129.37 (C-8); 131.30 (C-10); 135.79 (C-1a); 137.27 (C-8a);
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-methoxyphenyl)-4-thiazolidinone
(23)
Yellow solid. MW: 493.617. m.p. 130–1338C. Yield 59%, Rf ꢂ 100
value: 44.64 (M5). FT-IR (vmax, cmꢀ1): 3298 (indole and amide NH),
1705 (thiazolidinone, C O), 1668 (amide, C O). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.47–0.63 (m, 3H, –CH2–CH3
at C1); 1.04–2.23 (m, 7H, –CH2–CH3 at C1 and –CH2–CH3 at C8);
2.66–3.70 (m, 6H, –CH2–CO–NH at C1, –CH2 at C3 and C4) 3.75 (s,
3H, O–CH3); 3.79, 3.85 (2d and each 1H, –S–CH2–, J ¼ 16.7 and
16.0 Hz); 5.66, 5.75 (ss, 1H, –N–CH–S); 6.72–7.37 (m, 7H, Ar–H);
9.90, 9.98 (ss, 1H, indole NH); 10.30, 10.38 (ss, 1H, –CO–NH–N–).
13C NMR (75 MHz) (DMSO-d6/TMS): d (ppm): 8.30 (C-12); 14.94 (C-
10); 22.30 (C-9); 24.21 (C-4); 29.86 (C-11); 30.95 (thiazolidinone
S–CH2–); 42.42 (C-13); 55.68 (–OCH3); 60.52 (C-3); 62.03 (thiazoli-
dinone –S–CH–); 75.72 (C-1); 107.60 (C-4a); 114.34 (C-30 and C-50);
115.85 (C-6); 119.09 (C-5); 120.13 (C-7); 126.50 (C-8); 127.04 (C-20
and C-60); 129.79 (C-5a); 130.16 (C-10); 134.95 (C-1a); 136.50 (C-8a);
0
–
165.04 (C-4 ); 169.25 (CO–NH); 170.54 (thiazolidinone C O).
–
HR-MS (EIþ) Calcd./Found (m/z): 481.1835/481.1849 (Mþ)
(C26H28FN3O3S); 463.1930/463.1945; 450.2187/450.1684;
452.1439/452.1486 (C24H23FN3O3S); 435.1958/435.1467; 407.2009/
407.2008; 286.1681/286.1189; 270.1488/270.1443; 242.1539/
242.1193; 242.1175/242.1193; 240.1019/240.1042 (C15H14NO2);
229.1466/229.1404; 228.1382/228.1383 (C15H18NO); 227.1310/
227.1297; 212.1069/212.1032; 195.0518/195.0957.
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(2,6-difluorophenyl)-4-
thiazolidinone (21)
0
–
150.24 (C-4 ); 168.04 (CO–NH); 169.61 (thiazolidinone C O). HR-MS
–
(EIþ) Calcd./Found (m/z): 493.2035/493.2029 (Mþ) (C27H31N3O4S);
464.2007/464.2055 (C26H30N3O3S); 464.1638/464.1715; 463.1929/
463.1937 (C26H29N3O3S); 462.2393/463.1895; 419.2008/419.9784;
286.1681/286.1040; 270.1488/270.1422; 242.1539/242.1639;
242.1175/242.1185; 240.1019/240.1020 (C15H14NO2); 229.1466/
229.1398; 228.1382/228.1405 (C15H18NO); 227.1310/227.1319;
212.1069/212.1242; 210.0463/210.1187.
Yellow solid. MW: 499.573. m.p. 100–1028C. Yield 74%. Rf ꢂ 100
value: 33.33 (M2). FT-IR (vmax, cmꢀ1): 3348 (indole and amide NH),
1715 (thiazolidinone, C O), 1674 (amide, C O). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.51–1.40 (m, 6H, –CH2–CH3 at C1
and C8); 1.52–3.81 (m, 10H, –CH2–CH3 at C1 and C8; –CH2–CO–NH
at C1, –CH2 at C3 and C4); 3.85, 4.04 (2d and each 1H, –S–CH2–,
J ¼ 15.0 and 16.2 Hz); 6.06, 6.18 (ss, 1H, N–CH–S); 6.69–7.52 (m,
6H, Ar–H); 9.91, 10.0 (ss, 1H, indole NH); 10.30, 10.37 (ss, 1H,
–CO–NH–N–). 13C NMR (100 MHz) (DMSO-d6/TMS): d (ppm) 8.25 (C-
12); 14.98 (C-10); 22.38 (C-9); 24.31 (C-4); 29.96 (C-11); 31.49 (thia-
zolidinone S–CH2–); 42.18 (C-13); 60.52 (C-3); 60.91 (thiazolidi-
none –S–CH–); 76.08 (C-1); 108.35 (C-50 and C-30); 113.44 (C-4a);
116.57 (C-10); 119.92 (C-6); 120.87 (C-5); 127.19 (C-7); 127.26 (C-5a);
127.70 (C-40); 129.70 (C-8); 132.83 (C-1a); 135.78 (C-8a); 137.81 (C-20
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(2-chloro-6-fluorophenyl)-4-
thiazolidinone (24)
Yellow solid. MW: 516.027. m.p. 128–1308C. Yield 46%. Rf ꢂ 100
value: 32.14 (M2). FT-IR (vmax, cmꢀ1): 3343 (indole and amide NH),
1709 (thiazolidinone, C O), 1674 (amide, C O). 1H NMR
–
–
–
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
Etodolac Hydrazide Derivatives as Anticancer Agents
377
(400 MHz, DMSO-d6): d (ppm) 0.51, 0.56 (tt, 3H, –CH2–CH3 at C1);
0.84–2.74 (m, 9H, –CH2–CH3 at C1, –CH2–CH3 at C8, –CH2–CO–NH
at C1); 2.78–2.96 (m, 4H, –CH2 at C3 and C4); 3.76, 3.82 (2d and
each 1H, –S–CH2–, J ¼ 15.7 and 16.8 Hz); 6.28, 6.41 (ss, 1H,
–N–CH–S); 6.86–7.49 (m, 6H, Ar–H); 10.13, 10.27 (ss, 1H, indole
NH); 10.34, 10.38 (ss, 1H, –CO–NH–N–). HR-MS (EIþ) Calcd./Found
(m/z): 515.1445/515.1433 (Mþ) (C26H27ClFN3O3S); 497.1540/
497.1388; 486.1049/486.1020 (C26H27ClFN3O3S); 484.1798/
484.1220; 469.1568/469.0902; 463.1930/463.1859; 286.1681/
286.1214; 270.1488/270.1465; 246.0030/246.0021; 242.1539/
242.1559; 242.1175/242.1184; 240.1019/240.1037 (C15H14NO2);
229.1466/229.1413; 228.1382/228.1384 (C15H18NO); 227.1310/
227.1323; 212.1069/212.1207.
240.1019/240.1023 (C15H14NO2); 229.1466/229.1414; 228.1382/
228.1398 (C15H18NO); 227.1310/227.1307; 212.1069/212.1195;
195.0466/195.0536.
Anticancer activity
The primary anticancer assay was performed in accordance with
the protocol of the Drug Evaluation Branch, NCI (Bethesda, MD,
USA) [44–46, 52]. The human tumor cell lines of the cancer
screening panel were grown in RPMI 1640 medium containing
5% fetal bovine serum (FBS) and 2 mM L-glutamine. For a typical
screening experiment, 100 mL of cells was inoculated into 96-
well microtiter plates at plating densities ranging from 5000 to
40,000 cells/well, depending on the doubling time of the indi-
vidual cell lines. After cell inoculation, the microtiter plates were
incubated at 378C, 5% CO2, 95% air and 100% relative humidity
for 24 h prior to the addition of the experimental drugs. The
cytotoxic and/or growth inhibitory effects of the compounds
were tested in vitro against the full panel of 60 human tumor
cell lines derived from 9 neoplastic diseases, at 10-fold dilution.
The percentage of growth was evaluated spectrophotometrically
versus controls that were not treated with the test agents. Briefly,
the effects of the compounds on the growth parameters of the
different cancer cell lines were evaluated relative to controls
treated with equivalent amounts of DMSO, and expressed as
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-nitrophenyl)-4-thiazolidinone
(25)
Dark orange solid. MW: 508.589. m.p. 97–1008C. Yield 39%.
Rf ꢂ 100 value: 16.92 (M2). FT-IR (vmax, cmꢀ1): 3291 (indole and
–
–
–
amide NH), 1709 (thiazolidinone, C O), 1682 (amide, C O).
–
1H NMR (400 MHz, DMSO-d6):
d (ppm) 0.83–1.47 (m, 6H,
–CH2–CH3 at C1 and C8); 2.64–3.62 (m, 10H, –CH2–CH3 at C1
and C8; –CH2–CO–NH at C1; –CH2 at C3 and C4); 3.81, 3.87 (2d
and each 1H, –S–CH2–, J ¼ 16.1 and 17.3 Hz); 5.21–6.12 (m, 1H,
N–CH–S); 6.83–8.44 (m, 7H, Ar–H); 10.04–10.62 (m, 2H, indole NH
and –CO–NH–N–). 13C NMR (75 MHz) (DMSO-d6/TMS): d (ppm)
14.36 (C-12); 14.95 (C-10); 20.35 (C-9); 23.88 (C-4); 29.49 (C-11);
37.07 (thiazolidinone S–CH2–); 41.20 (C-13); 60.66 (C-3); 61.58
(thiazolidinone –S–CH–); 65.67 (C-1); 119.22 (C-4a); 119.95 (C-6);
123.66 (C-5); 124.30 (C-7); 126.77 (C-30 and C-50); 127.80 (C-8);
128.33 (C-20 and C-60); 128.70 (C-5a); 128.92 (C-1a); 129.35 (C-
percent growth rate. The compounds were added at 10ꢀ5
M
concentration for 48 h. Compounds 1, 9, 10, 20, and 21 chosen
as prototypes were evaluated against the full panel of 60 human
tumor cell lines at a single dose in the NCI in vitro primary
anticancer assay.
Cell viability studies (PC-3 cells)
8a); 147.50 (C-10); 169.17 (C-40); 169.69 (CO–NH); 171.02 (thiazoli-
Cell viability effects of etodolac and 2-(1,8-diethyl-1,3,4,9-tetrahy-
dropyrano[3,4-b]indole-1-yl)-N-[(4-chlorophenyl)methylene]aceto-
hydrazide (compound 9) were evaluated in vitro using the MTT
colorimetric method against the prostate cancer cell line PC-3
and the rat fibroblast cell line L-929, at different doses [53, 54], at
the Marmara University Faculty of Pharmacy, Department of
Pharmaceutical Biotechnology. MTT is cleaved to formazan by
the ‘‘succinate-tetrazolium reductase’’ system (EC 1.3.99.1),
which belongs to the mitochondrial respiratory chain and is
active only in viable cells. The PC-3 prostate cancer cell line
(ATCC1 CRL-1435TM) was used for the determination of anti-
cancer activity. This cell was cultured in Dulbecco’s Modified
Eagle Medium (DMEM; Gibco, Invitrogen) supplemented with
10% FBS (Gibco, Invitrogen), 1% glutamine, and streptomycin-
penicillin.
Testing in vitro was done by the method of Woerdenbag et al.
[54] with minor modifications. The MTT metabolic assay was
carried out at the seeding density of 1 ꢂ 104 cells/well in 96-well
flat-bottom cell culture plates with 100 mL of opti-MEM
(Invitrogen, USA). Following 24 h of incubation at 378C, 5%
CO2, the medium was aspirated; the compounds were dissolved
in DMSO and diluted with medium before addition to the cell
cultures at the concentrations of 5 and 10 mg/mL. The cells were
incubated for 48 h at 378C, 5% CO2. After the incubation period,
10 mL of the MTT labeling reagent (final concentration 0.5 mg/mL
(Cell Proliferation Kit MTT; Roche, Germany)) was added to each
well. The samples were incubated for 4–12 h in a humidified
atmosphere (e.g., 378C, 5.0% CO2) and 100 mL of the solubilization
buffer was added into each well. The plate was allowed to stand
overnight in the incubator in a humidified atmosphere (e.g.,
þ
–
dinone C O). HR-MS (EI ) Calcd./Found (m/z): 508.1780/508.1800
–
(Mþ) (C26H28N4O5S); 479.1753/479.1775 (C25H27N4O4S); 477.2132/
477.1950; 463.1929/463.1432; 462.1903/462.1538; 435.2026/
435.9687; 286.1681/286.1230; 270.1488/270.1459; 242.1539/
242.1534; 242.1175/242.1149; 240.1019/240.1205; 239.0364/
239.0337; 229.1466/229.0887; 228.1382/228.1390; 227.1310/
227.1315; 212.1069/212.1318.
3-(2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-
yl)acetylhydrazono)-2-(4-pyridinyl)-4-thiazolidinone (26)
Orange solid. MW: 464.580. m.p. 101–1038C. Yield 15%. Rf ꢂ 100
value: 90 (M7). FT-IR (vmax, cmꢀ1): 3312 (indole and amide NH),
1713 (thiazolidinone, C O), 1668 (amide, C O). 1H NMR
–
–
–
–
(400 MHz, DMSO-d6): d (ppm) 0.51–0.68 (m, 3H, CH2–CH3 at C1);
0.95–2.97 (m, 13H, CH2–CH3 at C1; CH2–CH3 at C8; –CH2–CO–NH
at C1; –CH2 at C3 and C4); 3.94, 4.12 (2d and each 1H, –S–CH2–,
J ¼ 16.0 and 14.2 Hz); 5.14–5.81 (m, 1H, N–CH–S); 6.86–8.70 (m,
7H, Ar–H); 10.04–10.55 (m, 2H, indole NH and CO–NH–N).
13C NMR (75 MHz) (DMSO-d6/TMS): d (ppm) 8.33 (C-12); 14.90 (C-
10); 19.02 (C-9); 22.25 (C-4); 24.18 (C-11); 33.26 (thiazolidinone
S–CH2–); 51.07 (C-13); 56.51 (C-3); 61.49 (thiazolidinone –S–CH–);
75.74 (C-1); 115.90 (C-4a); 119.21 (C-6); 120.15 (C-5); 121.55 (C-7);
122.87 (C-20 and C-60); 123.32 (C-8); 126.44 (C-5a); 127.03 (C-1a);
134.96 (C-8a); 145.89 (C-10); 149.83 (C-30 and C-50); 168.82 (CO–NH);
þ
–
171.04 (thiazolidinone C O). HR-MS (EI ) Calcd./Found (m/z):
–
464.1882/464.1859 (Mþ) (C25H28N4O3S); 437.1773/437.2261;
435.1485/435.1473 (C23H23N4O3S); 433.2234/433.2105;
418.2004/418.9758; 390.2056/390.2208; 286.1681/286.1646;
270.1488/270.1566; 242.1539/242.1370; 242.1175/242.1148;
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

378
P. C¸ ıkla et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 367–379
378C, 5% CO2) and the formazan precipitates were then solubil-
ized. The absorbance of the formazan product was measured
spectrophotometrically at 550 and 690 nm. Statistical analyses
were done using the unpaired Student’s t-test using Prism 3.0
(GraphPad Software, San Diego, CA, USA).
formaldehyde and subjected to spectrofluorimeter analysis (exci-
tation 490 nm, emission 520 nm). The results are presented as
RFU/mg protein.
Measurement of caspase-3 activity
Caspase-3 activity was determined using caspase colorimetric
assay kits (Sigma, St. Louis, MO, USA). In brief, cells were soni-
cated in ice-cold lysis buffer. Samples were centrifuged for
10 min at 10,000 ꢂ g at 48C. The supernatants were collected,
and the protein concentrations were measured by the Bradford
method. The caspase-3 assay was based on the spectrophotomet-
ric detection of cleavage of the chromophore p-nitroanilide (pNA)
from Ac-DEVD-pNA. Samples, in duplicate, were incubated for
1 h at 378C in a buffer mixture containing caspase-3 substrate
(Ac-DEVD-pNA) with or without the caspase-3 inhibitor (Ac-DEVD-
CHO). pNA (Sigma N2128) was used as the standard. Samples
were loaded into each well of a 96-well plate and incubated with
Ac-DEVD-pNA at 378C for 1 h. pNA cleavage was followed spec-
trophotometrically using an EPOCH microplate reader (Biotec,
USA) at 405 nm. The results were expressed as nanomoles of pNA
per minute per milligram of protein.
Apoptosis studies
The apoptosis pathway of etodolac and 2-(1,8-diethyl-1,3,4,9-
tetrahydropyrano[3,4-b]indole-1-yl)-N-[(4-chlorophenyl)methylene]-
acetohydrazide 9 was evaluated using the PC-3 prostatic cancer
cell line, at the Marmara University Faculty of Pharmacy,
Department of Biochemistry. Etodolac and compound 9 were
dissolved in DMSO and diluted in culture medium immediately
before use. The final concentration of DMSO in all experiments
was less than 0.01%, and all treatment conditions were compared
with vehicle controls. The cells were incubated with 100 mM
etodolac or 100 mM compound 9 for 24, 48, and 72 h at 378C.
Determination of the mitochondrial membrane potential by
spectrofluorimeter
Changes in the mitochondrial membrane potential were
measured according to the kit procedure (MitoPT JC-1 911)
using the J-aggregate-forming lipophilic cationic fluorochrome
JC-1. In healthy cells, the dye accumulates and aggregates
Statistical analysis
Data are presented as mean ꢄ SD. Data were analyzed using
repeated measures ANOVA followed by a post-hoc Tukey’s test
for multiple comparisons. A p-value of less than 0.05 was con-
sidered statistically significant.
in mitochondria, emitting
a bright red fluorescence. In
apoptotic and necrotic cells with altered mitochondrial mem-
brane potential, the dye remains in the cytoplasm in its mono-
meric form and the fluorescence is green. Thus, at low
membrane potential, JC-1 exists mainly in the green-fluorescent
monomeric form.
This research was supported by The Scientific and Technical Research
¨ ˙
Council of Turkey (TUBITAK), Research Fund Project Number: SBAG-
HYD-339 (108S257). The authors are grateful to Dr. Ju¨rgen Gross from
the Institute of Organic Chemistry, University of Heidelberg, for his
generous help on obtaining HR-EI/FAB mass spectra of the synthesized
compounds. We thank the Division of Cancer Research, National Cancer
Institute, Bethesda, MD, USA, for the anticancer activity screening.
Etodolac was supplied by Bilim Pharmaceutical Industry Inc.
Briefly, JC-1 was dissolved in 500 mL DMSO for preparing the
stock solution and then diluted in phosphate buffered saline (PBS)
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4,
pH 7.4) on the day of staining. First, cells (1 ꢂ 104 cells/well) were
incubated in the dark for 30 min at 378C in the JC-1 working
solution. Then, 5 mL JC-1 was added to the tubes and incubated for
20 min in the dark at 378C. The cells were washed twice with PBS
buffer. JC-1-loaded cells were excited at 490 nm, and emission was
detected at 510–570 nm (JC-1 aggregates) and 516 nm (JC-1 mono-
mers) using a spectrofluorimeter (GloMax; Promega, USA). Data are
presented as relative fluorescence units (RFU).
The authors have declared no conflict of interest.
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