Synthesis of Tolmetin Hydrazide-Hydrazones and Discovery of a Potent Apoptosis Inducer in Colon Cancer Cells

Article abstract of DOI:10.1002/ardp.201500178

Tolmetin hydrazide and a novel series of tolmetin hydrazide-hydrazones 4a-l were synthesized in this study. The structures of the new compounds were determined by spectral (FT-IR, ¹H NMR) methods. N′-[(2,6-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4g) was evaluated in vitro using the MTT colorimetric method against the colon cancer cell lines HCT-116 (ATCC, CCL-247) and HT-29 (ATCC, HTB-38) to determine growth inhibition and cell viability at different doses. Compound 4g exhibited anti-cancer activity with an IC₅₀ value of 76 μM against colon cancer line HT-29 (ATCC, HTB-38) and did not display cytotoxicity toward control NIH3T3 mouse embryonic fibroblast cells compared to tolmetin. In addition, this compound was evaluated for caspase-3, caspase-8, caspase-9, and annexin-V activation in the apoptotic pathway, which plays a key role in the treatment of cancer. We demonstrated that the anti-cancer activity of this compound was due to the activation of caspase-8 and caspase-9 involved in the apoptotic pathway. In addition, in this study, we investigated the catalytical effect of COX on the HT-29 cancer line, the apoptotic mechanism, and the moleculer binding of tolmetin and compound 4g on the COX enzyme active site. The tolmetin hydrazone N′-[(2,6-dichlorophenyl)methylidene]-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4g) exhibited anticancer activity with an IC₅₀ value of 76 μM against HT-29 cells and did not display cytotoxicity toward control fibroblast cells, compared to tolmetin. The anti-cancer activity of 4g was shown to be due to the activation of caspase-8 and caspase-9 involved in apoptosis.

Full text of DOI:10.1002/ardp.201500178

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
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Full Paper
Synthesis of Tolmetin Hydrazide–Hydrazones and Discovery of
a Potent Apoptosis Inducer in Colon Cancer Cells
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S¸. Guniz Kuc¸ukguzel , Derya Koc¸ , Pelin C¸ıkla-Suzgun , Derya Ozsavcı , Ozlem Bingol-Ozakpınar ,
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Pınar Mega-Tiber , Oya Orun , Pınar Erzincan , Safiye Sag-Erdem , and Fikrettin S¸ahin
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Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpas¸a,
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Istanbul, Turkey
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3
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Department of Biochemistry, Faculty of Pharmacy, Marmara University, Haydarpas¸a, Istanbul, Turkey
Department of Biophysics, School of Medicine, Marmara University, Bas¸ıbuyuk, Istanbul, Turkey
Department of Chemistry, Faculty of Arts and Sciences, Marmara University, Goztepe, Istanbul, Turkey
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Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe
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University, Kayıs¸dagı, Istanbul, Turkey
Tolmetin hydrazide and a novel series of tolmetin hydrazide–hydrazones 4a–l were synthesized in this
1
study. The structures of the new compounds were determined by spectral (FT-IR, H NMR) methods.
N0-[(2,6-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide
(4g) was evaluated in vitro using the MTT colorimetric method against the colon cancer cell lines
HCT-116 (ATCC, CCL-247) and HT-29 (ATCC, HTB-38) to determine growth inhibition and cell viability
at different doses. Compound 4g exhibited anti-cancer activity with an IC50 value of 76 mM against
colon cancer line HT-29 (ATCC, HTB-38) and did not display cytotoxicity toward control NIH3T3 mouse
embryonic fibroblast cells compared to tolmetin. In addition, this compound was evaluated for
caspase-3, caspase-8, caspase-9, and annexin-V activation in the apoptotic pathway, which plays a key
role in the treatment of cancer. We demonstrated that the anti-cancer activity of this compound was
due to the activation of caspase-8 and caspase-9 involved in the apoptotic pathway. In addition, in
this study, we investigated the catalytical effect of COX on the HT-29 cancer line, the apoptotic
mechanism, and the moleculer binding of tolmetin and compound 4g on the COX enzyme active site.
Keywords: Apoptosis / Caspase-3 / Colon cancer / Hydrazide–hydrazone / Tolmetin
Received: May 26, 2015; Revised: July 7, 2015; Accepted: July 21, 2015
DOI 10.1002/ardp.201500178
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
survival rate under 50%, despite the chemotherapy [1]. The
high prevalence and mortality of colorectal cancer make the
Introduction
Colorectal cancer is considered as the fourth leading cause of
cancer deaths. The worldwide incidence of colorectal cancer is
almost one million new malignant cases each year with a
search for effective prevention and/or treatments an urgent
medical concern. The use of chemopreventive approaches to
control colon cancer is an attractive strategy. The relationship
between inflammation and cancer is well established that
€
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Correspondence: Prof. S¸. Guniz Kuc¸ukguzel, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara
_
University, Haydarpas¸a 34668, Istanbul, Turkey.
This work was partly presented at the 4th International Meeting
on Pharmacy and Pharmaceutical Sciences, Istanbul, Turkey, 18–
23 September 2014.
E-mail: gkucukguzel@marmara.edu.tr
Fax: þ90-216-3452952
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
Tolmetin Hydrazones Against Colon Cancer
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suggests the use of anti-inflammatory drugs for the preven-
tion of colorectal cancer.
drawing attention in the scientific community due to their
several biological and clinical applications [12, 14, 19–21].
Activity of hydrazones is known to be associated with the
Non-steroidal anti-inflammatory drugs (NSAIDs) are a well-
known class of anti-inflammatory drugs that inhibit the
cyclooxygenase (COX) enzymes which are crucial for the
production of eicosanoids from arachidonic acid [2]. Reports
have shown the ability of many anti-inflammatory agents,
especially the NSAIDs, to inhibit tumor growth. Moreover,
several studies have revealed a substantial decrease in the
mortality from colorectal cancer in association with the use of
aspirin and other NSAIDs [3–5]. The mechanism of action that
is responsible for the chemopreventive activity of NSAIDs is
widely attributed to COX-2 inhibition [6–8] and a COX-2-
independent mechanism [9–11]. In addition, tumor growth
inhibition and apoptotic effect of the some compounds
derived from NSAIDs has been reported [12–16] (Fig. 1).
Tolmetin, 2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-
acetic acid, is a non-steroidal anti-inflammatory drug which
inhibits prostaglandin synthesis and it has been reported to
preventproliferationofcoloncancercells.Inaddition,tolmetin’s
regulatory effects on anti-cancer drug therapy were studied. It
was reported that tolmetin and other NSAIDs have effects on
increasing the cytotoxic activity of the anti-cancer drugs [17];
they inhibit the b-catenin functions and thatis whytolmetin can
be used for developing new anti-cancer agents [18].
–
active pharmacophoric group (–CONH–N C<). In the light of
–
these knowledge, 12 compounds which possess hydrazide–
hydrazone as main structure have been synthesized in
this study starting from tolmetin. 2-[1-Methyl-5-(4-methyl-
benzoyl)-1H-pyrrol-2-yl]-N0-[(pyridinyl/substitutedphenyl/2-
furyl)methylidene]acetohydrazides (4a–l) were synthesized.
Tolmetin (1) and tolmetin hydrazide–hydrazone 4g were
tested for their growth inhibitory and apoptotic activities
against colorectal tumor cells.
Results and discussion
Chemistry
Twelve novel hydrazide–hydrazones have been synthesized
from tolmetin hydrazide, which is a derivative of 2-[1-methyl-
5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetic acid (tolmetin); an
inhibitor of prostaglandine synthesis and a non-steroidal anti-
inflammatory drug. Structures of the synthesized compounds
were confirmed by UV, IR, 1H NMR and LC-MS (EI) (only 3)
and their purity was checked by elemental analysis, TLC, and
HPLC-diode array.
Hydrazide–hydrazones, which are intermediates in the
synthesis of biologically active compounds, are widely
As shown in the synthesis scheme, tolmetin was first
synthesized from tolmetin sodium dihydrate by hydrolyzing
Figure 1. Tumor growth inhibiting NSAIDs
(etodolac, celecoxib, diflunisal, flurbiprofen).
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the dihydrate in acidic conditions. Methyl 2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetate (2) has been synthe-
sized from tolmetin (1) with methanol and few drops of
concentrated sulfuric acid. 2-[1-Methyl-5-(4-methylbenzoyl)-
1H-pyrrol-2-yl]acetohydrazide (3) was synthesized from com-
pound 2, which is an ester, with hydrazine hydrate and
methanol. Compound 3, tolmetin hydrazide derivative, was
refluxedwithsubstituedaldehydesandledustothesynthesisof
2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N0-[(pyridinyl/
substitutedphenyl/2-furyl)methylidene]acetohydrazides (4a-l),
hydrazide–hydrazone compounds (Fig. 2).
pyrrol-2-yl]acetic acid (tolmetin) was reacted with methanol
in the presence of concentrated sulfuric acid, which leads
to the synthesis of methyl 2-[1-methyl-5-(4-methylbenzoyl)-
1H-pyrrol-2-yl]acetate (2). Compound 2 is not an original
compound [23]. FT-IR spectrum of compound 3 showed
ꢀ1
1720 cmꢀ1 (C O), 1205 cm
(C–O), both belong to ester
–
–
functional group. Besides, 3370 cmꢀ1 (O–H, carboxylic acid)
disappeared. 1H NMR spectrum of tolmetin ester was
determined at 3.81 ppm (3H, s, COOCH3) and there was no
peak in the spectrum which belongs to OH proton of caboxylic
acid group; which lead us to conclude that ester form of
tolmetin was succesfully synthesized. Compound 3 is an
original compound which was synthesized from methyl 2-[1-
methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetate (2) in the
presence of hydrazine–hydrate and methanol under reflux
for 3 h and the yield was 84%. The UV spectrum of tolmetin
hydrazide (3) established two maximum absorbance values at
254 and 300 nm in ethanol. The FT-IR spectrum of compound 3
Tolmetin sodium dihydrate was dissolved in water and
37% HCl was added until it stopped precipitating. The white
precipitate was controlled with pH paper. Tolmetin yield was
95%. FT-IR spectrum data of 2-[1-methyl-5-(4-methylbenzoyl)-
1H-pyrrol-2-yl]acetic acid (1) were observed 1688 cmꢀ1(C O),
–
–
3370 cmꢀ1 (O–H), and 1225 cmꢀ1 (C–O) from carboxylic acid
functional group [22]. 2-[1-Methyl-5-(4-methylbenzoyl)-1H-
Figure 2. Synthetic route of tolmetin hydrazide–hydrazones (4a–l).
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showed 3300 cmꢀ1 (CONHNH2), which proves the hydrogen
compound 4g using MTT colorimetric assay [27] against panel
of human colon cancer cell lines. Tolmetin hydrazone (4g) was
found to be active on HT-29 cell line with 76 mM of IC50 value.
Furthermore, tolmetin hydrazide–hydrazone was found to
be more active on HT-29 cancer cell line in higher doses as
shown in Table 1 and Fig. 3.
It was acknowledged that cancer cells resist to the normal
death pathways. Cancer cells use different mechanisms
including extrinsic and intrinsic pathways to escape apoptosis.
The evidence obtained in recent years indicates that many
non-steroidal anti-inflammatory drugs (NSAIDs) have anti-
proliferative activity in tumor cells [28, 29]. In the current
study, we also investigated whether the anti-cancer effects
of compound 4g and tolmetin occurred via the apoptotic
pathway and, if so, which signaling pathway that initiates
apoptosis was activated in host defence.
bond in the compound and 1643 cmꢀ1 (C O, hydrazide) in
–
–
compound 3. 1H NMR spectrum of compound 3 in DMSO-d6
showed that methyl protons disappeared at 3.81 ppm (3H, s,
COOCH3). 1H NMR spectrum of tolmetin hydrazide was
observed at 4.27 ppm (1H, s, NH2) and at 9.23 ppm (1H, s,
NH). This information showed us that ester derivative was
transformed to hydrazide derivative and –CONHNH2 group
was formed. Mass spectra of tolmetin hydrazide (3) with
electron impact method were determined at m/z 271.1 as
molecular ion peak and m/z 212.1 as base peak (100%).
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide
(3) and substituted aldehydes were refluxed in the presence
of ethanol for 2 h which led to the synthesis of hydrazide–
hydrazone compounds 4a–l, yield 50–93%. The FT-IR spectra of
(4a–l) compounds showed 3069–3231 cmꢀ1 (hydrazone, N–H).
–
–
The C O stretch band in hydrazone CONHN CH structure
Early stages of apoptosis are characterized by phosphati-
dylserine (PS) externalization on the outer surface of the
plasma membrane [30]. To determine the effects of tolmetin
and compound 4g depending on doses on PS exposure, we
used annexin V staining. According to our results, tolmetin
and 4g (SGK-417) increased PS externalization as compared
with the control group but this increase was significantly
greater for 4g than tolmetin in 100 mM (p < 0.001) (Fig. 4).
After the flip floating of PS, apoptotic death signaling
triggers and activates caspases. Caspases are known as
–
–
–
–
(amide I band) and C N stretch band were observed at 1660–
1692 and 1597–1628 cmꢀ1, respectively. The low cmꢀ1 N–H
stretch bands were observed in some compounds, which lead
ustotheconclusionthatsomeoftheN–Hgroupshadhydrogen
bonds. 1H NMR spectra of the compounds 4b–l at 300 MHz
–
–
frequency in CDCl , N–H protons, and azomethine (CH N)
3
protons were detected at 9.58–11.43 ppm (1H, s) and 7.79–
8.20 ppm (1H, s), respectively. Besides that, pyrrole N–CH3 (3H,
s) and CH2 protons were detected at 3.84–4.05 ppm and at
4.08–4.23 ppm (2H, s). The 1H NMR spectra of tolmetin 2-furyl
derivative (4a) were studied in DMSO-d6 and hydrazone N–H
–
Table 1. Percentage of concentration-growth inhibition
against HT-29 cell line.
proton was seen at 11.42–11.67 ppm (1H, s); CH N proton was
–
seen at 8.07–8.21 ppm (1H, s), respectively. In the light of
foregoing and according to the literature [14, 24, 25], we
assume that our compound chose the E isomer form as N–H
protonsweredetected at11.42and11.67 ppm. Thetwosinglet
peakswereobservedbecauseofthecis/transconformersofthe
Growth inhibition (%)
Concentration
Tolmetin
4g (SGK 417)
10ꢀ6
10ꢀ5
10ꢀ4
10ꢀ3
10ꢀ2
17.58
29.23
34.15
37.76
41.62
21.3
34.57
50.66
62.99
70.23
1
resonance form of the E isomer compound’s amide. H NMR
spectra of the same compound showed that hydrazone N–H
–
proton and CH N proton were observed at 8.84 ppm (1H, s)
–
and 7.65 ppm (1H, s), respectively. 2-Furfuryl derivative (4a)
was studied by 1H NMR at 600 MHz in DMSO-d6 and the pyrole
N–CH3 peak was overlapped in the solution peak. As a result,
its spectra were studied again in CHCl3 and N–CH3 peak was
observed in 4.04 ppm.
Biological activity
In the present day, it is reported that tolmetin inhibits the
growth of colon cancer cells [26]. On the light of foregoing,
we aimed to investigate the anti-cancer activity of tolmetin
hydrazide–hydrazone 4g, which is an original compound and
a derivative of tolmetin, a substance with reported anti-
cancer activities human colon cancer cell lines, HCT-116 (ATCC,
CCL-247) and HT-29 (ATCC, HTB-38) were used to investigate
the anti-cancer effects of the compound. These studies were
carried out at Department of Biochemistry, Faculty of
Pharmacy, Marmara University. The evaluation of anti-cancer
and cytotoxicity effects was performed by measuring the
levels of alive cells after incubation with the tolmetin and
Figure 3. Cytotoxic activity of tolmetin and compound 4g (SGK-
417) on HT-29 cells.
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Figure 4. The early apoptotic effects of tolmetin and compound
4g (SGK-417) on HT-29 cells. Data shown as mean ꢂ SD in
triplicate culture and a representative of two independent
experiments. ꢃp < 0.05; ꢃꢃp < 0.001; ꢃꢃꢃp < 0.0001 versus control;
þp < 0.0001 versus SGK-417.
Figure 6. The effects of tolmetin and compound 4g (SGK-417)
on caspase 8 activity in HT-29 cells. Data shown as mean ꢂ SD
in triplicate culture and a representative of two independent
experiments. ꢃp < 0.05; ꢃꢃꢃp < 0.0001; gp < 0.01 versus control;
þþp < 0.05 versus SGK-417.
cysteine-proteases and play an important role in apoptosis.
They normally exist in a precursor form that has little activity
but once activated, breakdown structural elements and other
proteins. Caspases 8 and 9 are known as initiator and caspase
3 is an effector protein in the apoptotic signaling pathways.
Caspase 9 is predominantly defined in the intrinsic pathway
and caspase 8 is described in the extrinsic pathway. Depend-
ing on the cell type and stimulus, the cells determine which
caspases are used. Both pathways generally overlap down-
stream with the activation of caspases 3, 6, and 7 [31, 32].
Caspases 3, 8, and 9 enzyme levels were significantly
increased in the colon cancer cells which were incubated with
tolmetin (1 (100 mM) and 4g (SGK-417)) (Figs. 5–7). Loss of
mitochondrial membrane potential is a hallmark of intrinsic
apoptotic pathway. In this study, we also observed the effects
of tolmetin and compound 4g on the mitochondrial
membrane potential as shown in Fig. 8, JC-1 fluorochrome
staining. Moreover, we showed that both compounds
Figure 7. The effects of tolmetin and compound 4g (SGK-417)
on caspase 9 activity in HT-29 cells. Data shown as mean ꢂ SD
in triplicate culture and a representative of two independent
experiments. ꢃp < 0.05; ꢃꢃꢃp < 0.0001 versus control.
induced disturbance of the mitochondrial membrane poten-
tial and activation of caspases 3, 8, and 9, all of which are
involved in extrinsic and intrinsic apoptotic pathways.
COX-2 is induced in many inflammatory reactions. Recent
studies report that COX-2 expression is abnormally elevated
in some colon adenomas [33]. Various animal and human
tumor tissues, including human colon cancer, contain high
concentrations of prostaglandins. Therefore, these observa-
tions led us to investigate catalytical effects of COX enzymes
on HT-29 cancer line.
Inhibitory action of tolmetin and 4g was first observed
using COX-1 standard provided by the kit. COX-1 inhibitor
SC-560 and tolmetin inhibited COX-1 activity as expected.
Total COX activity was very low in HT-29 cells (just above the
basal level) for approximately 8 ꢁ 105 cells, where activity of
MCF-7 cells were 1.3-fold higher with a half cell population
(4 ꢁ 105 cells). There was no detectable change in the COX-
activities after inhibitors were added, but the number of HT-
29 cells was considerably reduced (Table 2). These results
Figure 5. The effects of tolmetin and compound 4g (SGK-417)
on caspase 3 activity in HT-29 cells. Data shown as mean ꢂ SD in
triplicate culture and a representative of two independent
experiments. ꢃp < 0.05; ꢃꢃp < 0.001; ꢃꢃꢃp < 0.0001; gp < 0.01
versus control; þp < 0.0001 versus 4g.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
Tolmetin Hydrazones Against Colon Cancer
Archiv der Pharmazie
Figure 8. Fluorescence microscopy after JC-1 staining showing changes in the mitochondrial membrane potential in HT-29 cells;
magnification: ꢁ100).
encouraged us to investigate molecular binding of tolmetin
and 4g with COX enzyme active site.
binding mechanism. COX enzymes have two different
catalytic sites: Cyclooxygenase active site is a hydrophobic
channel elongated toward the membrane domain. It is the
target of NSAID drugs. Peroxidase active site is situated at the
surface of catalytic domain and contains a heme cofactor.
Although the nature of substrate binding to cyclooxygenase
site is well established, peroxidase site is less explored.
In silico studies
COX-1 and COX-2 binding affinities of tolmetin and 4g were
explored with molecular docking using AutoDock Vina
software [34] in order to get insight into the inhibitory
Table 2. COX activities and effect of inhibitors were determined both in isolated ovine COX-1 standard and two cancer
cell lines, HT-29 and MCF-7.
Total activity
nmol/min/mL
% Decrease in
Cox-activity
% Decrease in cell number
with respect to control
COX-1 standard
COX-1 Standard
Std þ DuP-697 (COX2 inh)
Std þ SC-560 (COX1 inh)
Tolmetin
23.20
13.67
5.02
3.56
19.70
–
9.53
18.18
19.64
3.50
4g
Cancer cell lines
HT-29-control
HT-29 þ Tolmetin
HT-29 þ 4g
6.04
6.17
6.42
–
–
0.00
ꢀ0.25
55
60
MCF7-control
MCF7 þ Tolmetin
MCF7 þ 4g
8.45
8.33
8.90
–
–
41.1
0
0.25
ꢀ0.32
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However, it is known that both sites are involved in catalytic
reaction [35].
tolmetin and alkyl groups of Leu531 and Leu352 residues
similar to the reference inhibitor. Additionally, aromatic
p-electrons of Trp387 and Tyr385 interact favorably with para
CH3 substituent in tolmetin. Similar to tolmetin, 4g also fits
cyclooxygenase active site mainly with hydrophobic inter-
actions with Leu384, Leu352, and Ala527. Dichlorophenyl
group of 4g is surrounded by Ile523, Tyr355, Leu93, Ile89,
and Val116 and exhibits p-cation interactions with Arg120
which is critical for slightly more favorable binding of 4g
with respect to tolmetin where dichlorophenyl moeity is
absent.
According to AutoDock Vina docking scores, both tolmetin
and 4g are binded to COX-1 cyclooxygenase active site as well
as to peroxidase active site. Binding energies for tolmetin and
4g at COX-1 cyclooxygenase active site were calculated as
ꢀ8.1 and ꢀ8.6 kcal/mol, respectively. Although 4g exhibited
slightly more binding affinity than tolmetin (elucidated by its
smaller free energy of binding), these compounds showed less
binding affinity to the cyclooxygenase active site of COX-1
enzyme relative to the reference pdb inhibitor a-methyl-4-
biphenylacetic acid (the defluorinated analog of flurbiprofen)
whose binding energy was calculated as ꢀ9.1 kcal/mol. These
results are consistent with biological activity measurement.
Binding mode of reference pdb inhibitor showed favorable
p-alkyl interactions between its phenyl moiety and Leu352
and Leu531 residues in addition to H-bonding to Arg120.
Binding orientations of tolmetin and 4g in COX-1 are depicted
in Fig. 9.
As stated before, some conformers of tolmetin and 4g
also bind to peroxidase active site of COX-1. In the literature,
there are several docking studies for various inhibitors binding
to cyclooxygenase active site of COX enzymes [36]. However,
as far as we know, peroxidase active site is less explored
and inhibitors binding to peroxidase active site are not yet
reported. According to AutoDock Vina scores, tometin and 4g
bound more favorably to peroxidase site than cyclooxygenase
site of COX-1. The binding affinity of 4g was even stronger
than tolmetin as observed from the calculated binding
energies of ꢀ9.7 and ꢀ8.8 kcal/mol, respectively.
Figure 9 reveals that cyclooxygenase binding mechanisms
of both compounds are dominated by hydrophobic inter-
actions except one non-bonded attraction between –CH3
hydrogen of pyrrole ring in tolmetin and –OH oxygen of
Tyr355. Other favorable interactions responsible for binding
of tolmetin are p-alkyl interactions between aromatic rings of
Binding orientation of tolmetin and 4g in peroxidase
active site of COX-1 are illustrated in Fig. 10. Both tolmetin
and 4g exhibit favorable p–p stacking interactions with
Figure 9. Binding orientation of tolmetin (left: pink) and 4g (right: yellow) in the cyclooxygenase active site of COX-1. Interaction
̊
distances are given in A.
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Tolmetin Hydrazones Against Colon Cancer
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Figure 10. Binding orientation of tolmetin (left: pink) and 4g (right: yellow) in the peroxidase active site of COX-1. Interaction
̊
distances are given in A.
porphyrin ring of heme cofactor and p–alkyl interactions
with His207 which is essential for peroxidase activity of
COX-1. Additional interactions of 4g responsible for its
higher affinity are the strong H-bond with His274 (2.78 A),
and the interactions of chlorophenyl moiety with Leu408 and
His446.
Tolmetin is known to be a selective COX1 inhibitor. In
accordance with this, only a few conformers of tolmetin
bound to COX-2 with the binding energy of ꢀ6.6 kcal/mol
revealing a negligibly weak affinity. On the other hand, none
of the conformers of 4g exhibited binding to COX-2 indicating
its higher selectivity to COX-1 with respect to tolmetin.
which was also reported to be active in colon cancer and
compound 4g for their anti-cancer activity at Department of
Biochemistry, Marmara University. Therefore, whether hydra-
zone functionality increases the anti-cancer activity or not
comparing to tolmetin was studied. According to MTT assays,
compound 4g was found to be more active than tolmetin only
in HT-29 cancer cell line, in a dose-dependent manner with
IC50 value of 76 mM.
Caspase-3 is an enzyme catalyzing specific cell key protein
division and is mostly induced fatal protease. This activation
starts with caspase-8 and caspase-9 shred. Caspase-8 is a basic
compound for exogenous cell death pathway by TNF family
members. Caspase-9 is a key compound for mitochondrial
(endogenous) pathway. Caspase-9 activates the other cas-
pases. To evaluate the apoptotic mechanisms which are
important in cancer formation, caspase activities were studied
in HT-29 cell line (according to MTT assay) and the changes in
the levels of caspase 3, 8, and 9 were observed. For this study,
75, 100, and 150 mM concentrations of compounds 1 and 4g
were used. Annexin V, which is a protein existing on the
surface of the cells that undergo apoptosis, was also used to
determine early apoptosis in the cells [39].
̊
Conclusion
One of the beneficial methods of synthesizing a pro-drug is to
hydrolyze the hydrazones and reveal its active metabolite,
therefore, we can block the –NH2 group and have compounds
that are less toxic than hydrazides [37]. In the recent studies,
researchers focus on synthesizing more active, less toxic
chemotherapeutics and this led them to the synthesis of new
hydrazide–hydrazones compounds. Both the anti-cancer
activity and the apoptotic effects of these molecules are
popular studies [38].
In the light of foregoing, compound 4g, bearing hydra-
zone functionality, triggers apoptosis by affecting caspase 3
levels and does that by using caspase 8 and caspase 9
pathways in cancer cells. It is also observed that HT-29 colon
cancer cells which are treated with 4g and undergo
HCT-116 (ATCC, CCL-247) and HT-29 (ATCC, HTB-38) human
colon cancer cell lines are used for the studies of tolmetin (1)
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apoptosis, have high levels of annexin V with the dose of 75–
100 mM.
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetic
acid (1)
We believe that anti-colon cancer activity will exist in
tolmetin chromophore group as a result of the HT-29 anti-
cancerogenic activity of both tolmetin and tolmetin hydra-
zone 4g, which activates caspases 3, 8, and 9; effective
enzymes in apoptosis pathway.
Tolmetin sodium dihydrate (0.01 mol) was dissolved in
distilled water (22 mL). HCl (37%) was added on the resulting
solution until completion of the deposition process, and was
controlled with litmus paper blue. The resulting white
precipitate was filtered, washed with distilled water, dried,
and crystallized from ethanol. MW: 257.284. m.p. 157°C.
Yield 95%. Rf ꢁ 100 value 70 (M1). HPLC tR (min): 1.006. IR
Docking calculations predicted compound 4g as
a
selective inhibitor of COX-1. Despite its smaller affinity
to cyclooxygenase site of the enzyme, it binds to per-
oxidase site more favorably. Owing to this ability, it is
expected to have a different mode of action than other
NSAIDs drugs. It can be a potent novel therapeutic agent—
by inducing apoptosis in HT-29 cells through activation of
caspases and that this activation involves COX-1-mediated
mechanisms.
(vmax, cmꢀ1): 3370 (carboxylic acid OH), 1688 (C O).
–
–
Methyl 2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-
acetate (2)
Tolmetin (0.01 mol) and methanol (20 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 recrystallized twice
from ethanol. Cream-colored solid. MW: 271.311. m.p. 121–
123°C [35]. Yield 77%. Rf ꢁ 100 value 32 (M2). HPLC tR (min):
3.369. UV (EtOH) ʎmax nm: 318, 311, 299, 256. IR
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
compounds were determined in a Schmelzpunktbestimmer
SMPII melting point apparatus and are uncorrected. The purity
of the compounds was checked on TLC plates pre-coated with
silica gel G using the solvent systems M1 (chloroform/methanol
40:60 v/v), M2 (petroleum ether/ethyl acetate 50:50 v/v), and M3
(petroleum ether/ethyl acetate 30:70 v/v). The spots were
located under UV light (254 nm) (T ¼ 21°C). The liquid chro-
matographic system consists of an Agilent Technologies 1100
series instrument equipped with a quaternary solvent delivery
system and a model Agilent series G1315 A photodiode array
detector.ARheodynesyringeloadingsampleinjectorwitha50-
mL sample loop was used for the injection of the analytes.
Chromatographic data were collected and processed using
Agilent Chemstation Plus software. The separation was
performed at ambient temperature using a normal phase
Ace 3 C18 (10cm ꢁ 4.0 mm). All experiments were performed in
gradient mode. The mobile phase was prepared by mixing
acetonitrile/distilled water (50:50) (v/v) for all compounds
filtered through a 0.45-mm pore filter and subsequently
degassed by ultrasonication, prior to use. Solvent delivery
was employed at a flow rate of 1 mL/min. Detection of the
analytes was carried out at 254 nm. Elemental analyses were
performed on a CHNS-932 (LECO). FT-IR spectra were recorded
on a ShimadzuFT-IR-8400S spectrophotometer. 1H NMR spectra
were recorded on Bruker 300 MHz Ultrashield TM and Bruker
600 MHz UltrashieldPlus TM (300 MHz) NMR spectrometers
using CDCl3 as solvent. MALDI-TOF HR-MS spectra using the EI
ionization techniques were performed using a Jeol JMS-700
instrument. Chemical shifts (d) are reported in parts per million
(ppm). Data are reported as follows: chemical shift, multiplicity
(s: singlet, d: doublet, m: multiplet, and t: triplet), coupling
constants (Hz), and integration.
(vmax, cmꢀ1): 1720 (ester C O), 1205 (ester C–O). 1H NMR
(300 MHz, DMSO-d6):
d (ppm) 2.49 (3H, s, Ar-CH3);
3.66 (3H, s, N–CH3); 3.81 (3H, s, O–CH3); 3.90 (2H, s,
–CH2–); 6.11 (1H, d, pyrrole ring protons adjacent CH2,
J ¼ 3.9 Hz); 6.56 (1H, d, pyrrole ring protons adjacent
–
C O, J ¼ 3.9 Hz); 7.29 (2H, d, benzene ring o-protons by
–
methyl, J ¼ 7.81 Hz); 7.29 (2H, d, benzene ring o-protons by
methyl, J ¼ 7.81 Hz); 7.62 (2H, d, benzene ring o-protons
by carbonyl, J ¼ 7.81 Hz).
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-
acetohydrazide (3)
To a methanolic solution of compound 2 (50 mL, 0.01 mol) was
added hydrazine–hydrate (80%, 5 mL) and refluxed for 3 h.
The reaction mixture was then cooled. The precipitated solid
was washed with water, dried, and recrystallized twice from
ethanol. Cream-colored solid. MW: 271.31438. m.p. 185–
186°C. Yield 84%. Rf ꢁ 100 value 24.5 (M3). HPLC tR (min):
1.093. UV (EtOH) ʎmax nm: 320, 312, 300, 254. FT-IR
–
(vmax
,
cmꢀ1): 3329, 3254 (hydrazide NH), 1643 (C O).
–
1H NMR (300 MHz, DMSO-d6): d (ppm) 2.37 (3H, s, Ar-CH3);
3.52 (2H, s, –CH2–CO–); 3.86 (3H, s, N–CH3); 4.27 (2H, s, –NH2);
6.06 (1H, d, pyrrole ring protons adjacent CH2, J ¼ 3.9 Hz);
–
–
6.54 (1H, d, pyrrole ring protons adjacent C O, J ¼ 3.9 Hz);
7.29 (2H, d, benzene ring o-protons by methyl, J ¼ 7.81 Hz);
7.61 (2H, d, benzene ring o-protons by carbonyl, J ¼ 8.3 Hz);
9.23 (1H, s, –NH). MS [EI, m/z, (%)]: 271.1 [Mþ] (% 62), 240.1
(% 18), 212.1 (% 100), 119 (% 27), 91 (% 14).
General procedure for the synthesis of 2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]-N0-[(pyridinyl/substituted
phenyl/2-furyl)methylidene]acetohydrazides (4a–l)
A solution of compound 3 (0.0025 mol) was dissolved in
ethanol (20 mL) and equimolar amounts of appropriate
aromatic aldehyde in absolute ethanol were heated under
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
Tolmetin Hydrazones Against Colon Cancer
Archiv der Pharmazie
–
reflux for 2 h. The obtained precipitate was filtered off, dried,
and recrystallized twice from ethanol.
J ¼ 4.05 Hz); 6.72 (1H, d, pyrrole ring protons adjacent C O,
–
–
–
J ¼ 4.05 Hz); 7.07–7.75 (8H, m, Ar–H); 7.84 (1H, s, –CH N); 10.10
(1H, s, –NH). Anal. calcd. for C22H20FN3O2.1/2C2H5OH: C, 68.98;
N0-[Furan-2-yl-methylidene]-2-[1-methyl-5-(4-
H, 5.79; N, 10.49%. Found: C, 69.04; H, 5.33; N, 11.33%.
mehylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4a)
Cream-colored solid. MW: 349.38316. m.p. 222–224°C. Yield
93%. Rf ꢁ 100 value: 28 (M2). HPLC tR (min): 2.732. FT-IR
N0-[(4-Chlorophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4e)
Cream-colored solid. MW: 416.900. m.p. 198–200°C. Yield
(vmax, cmꢀ1): 3183 (hydrazone NH), 1665 (C O), 1620 (C N).
–
–
–
–
1H NMR (600 MHz, DMSO-d6): d (ppm) 2.36 (3H, s, Ar-CH3); 3.84
(in solvent peak N–CH3); 4.07 (2H, s, –CH2–); 6.10–7.58 (9H, m,
protons of pyrrole and furan ring, Ar–H); 8.07, 8.21 (1H, ss,
70%. Rf ꢁ 100 value: 45 (M2). HPLC tR (min): 4.323. FT-IR (vmax
,
cmꢀ1): 3154 (hydrazone NH), 1684, 1666 (C O), 1597 (C N).
–
–
–
–
1H NMR (300 MHz, CDCl3): d (ppm) 1.67 (s, –CH3 peak of
ethanol held by the molecule); 2.44 (3H, s, Ar–CH3); 3.82
(s, –CH2 peak of ethanol that held by the molecule); 3.98
(s, –OH peak of ethanol that held by the molecule); 4.04
(3H, s, N–CH3); 4.21 (2H, s, –CH2–); 6.18 (1H, d, pyrrole ring
protons adjacent CH2, J ¼ 4.06 Hz); 6.71 (1H, d, pyrrole ring
–
–CH N); 11.42,11.67 (1H, ss, –NH). Anal. calcd. for C20H19N3O3:
–
C, 68.75; H, 5.48; N, 12.03%. Found: C, 68.24; H, 5.25;
N, 11.89%.
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N0-
[pyridin-3-yl-methylidene]acetohydrazide (4b)
Cream-colored solid. MW: 392.451. m.p. 191–193°C. Yield
–
protons adjacent C O, J ¼ 4.04 Hz); 7.24–7.75 (8H, m, Ar–H);
–
–
7.82 (1H, s, –CH N); 9.97 (1H, s, –NH). Anal. calcd. for
–
74%. Rf ꢁ 100 value: 17 (M2). HPLC tR (min): 2.007. FT-IR (vmax
,
C22H20ClN3O2.1/2C2H5OH: C, 66.26; H, 5.56; N, 10.08%. Found:
C, 65.74; H, 5.070; N, 10.51%.
1
cmꢀ1): 3069 (hydrazone NH), 1676 (C O), 1604 (C N). H NMR
–
–
–
–
(300 MHz, CDCl3): d (ppm) 1.78 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.83 (s, –CH2 peak of
ethanol that held by the molecule); 3.97 (s, –OH peak of
ethanol that held by the molecule); 4.04 (3H, s, N–CH3); 4.22
(2H, s, –CH2–); 6.19 (1H, d, pyrrole ring protons adjacent CH2,
N0-[(2,4-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4f)
Cream-colored solid. MW: 451.345. m.p. 202–206°C. Yield
82%. Rf ꢁ 100 value: 50 (M2). HPLC tR (min): 5.083. FT-IR (vmax
,
1
J ¼ 4.06 Hz); 6.71 (1H, d, pyrrole ring protons adjacent C O,
cmꢀ1): 3140 (hydrazone NH), 1692 (C O), 1597 (C N). H NMR
–
–
–
–
–
–
J ¼ 4.04 Hz); 7.24–7.74; 8.02–8.06 (8H, m, Ar–H); 7.89 (1H, s,
(300 MHz, CDCl3): d (ppm) 1.66 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.82 (s, –CH2 peak of
ethanol that held by the molecule); 3.98 (s, –OH peak of
ethanol that held by the molecule); 4.03 (3H, s, N–CH3); 4.19
(2H, s, –CH2–); 6.16 (1H, d, pyrrole ring protons adjacent CH2,
–
–CH N); 10.16 (1H, s, –NH). Anal. calcd. for C21H20N4O2.1/
–
2H2O.1/2C2H5OH: C, 67.33; H, 6.16; N, 14.28%. Found: C, 67.73;
H, 5.52; N, 15.17%.
N0-[(2-Fluorophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4c)
Cream-colored solid. MW: 400.445. m.p. 185°C. Yield 50%.
Rf ꢁ 100 value: 32 (M2). HPLC tR (min): 3.760. FT-IR (vmax, cmꢀ1):
J ¼ 4.06 Hz); 6.70 (1H, d, pyrrole ring protons adjacent C O,
–
–
–
–
J ¼ 4.05 Hz); 7.24–7.93 (7H, m, Ar–H); 8.20 (1H, s, –CH N); 9.81
(1H, s, –NH). Anal. calcd. for C22H19Cl2N3O2.1/2C2H5OH: C,
61.20; H, 4.91; N, 9.31%. Found: C, 60.75; H, 4.42; N, 9.65%.
3192 (hydrazone NH), 1674 (C O), 1628 (C N). 1H NMR
–
–
–
–
(300 MHz, CDCl3): d (ppm) 1.66 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.83 (s, –CH2 peak of
ethanol that held by the molecule); 3.99 (s, –OH peak of
ethanol that held by the molecule); 4.05 (3H, s, N–CH3); 4.21
(2H, s, –CH2–); 6.19 (1H, d, pyrrole ring protons adjacent CH2,
N0-[(2,6-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4g)
Cream-colored solid. MW: 428.311. m.p. 225–226°C. Yield
86%. Rf ꢁ 100 value: 60 (M2). HPLC tR (min): 4.683. UV (EtOH)
ʎmax nm: 310, 259, 220, 200. FT-IR (vmax
,
cmꢀ1): 3184
J ¼ 4.05 Hz); 6.71 (1H, d, pyrrole ring protons adjacent C O,
(hydrazone NH), 1660 (C O), 1622 (C N). 1H NMR
–
–
–
–
–
–
–
J ¼ 4.05 Hz); 7.10–7.44 (8H, m, Ar–H); 8.09 (1H, s, –CH N); 9.58
(300 MHz, CDCl3): d (ppm) 2.44 (3H, s, Ar–CH3); 4.01 (3H, s,
N–CH3); 4.20 (2H, s, –CH2–); 6.19 (1H, d, pyrrole ring protons
adjacent CH2, J ¼ 4.06 Hz); 6.70 (1H, d, pyrrole ring protons
–
(1H, s, –NH). Anal. calcd. for C22H20FN3O2.1/2C2H5OH: C, 68.98;
H, 5.79; N, 10.49%. Found: C, 69.33; H, 5.28; N, 11.03%.
–
adjacent C O, J ¼ 4.05 Hz); 7.24–7.74 (7H, m, Ar–H); 8.13 (1H, s,
–
N0-[(4-Fluorophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4d)
Cream-colored solid. MW: 400.445. m.p. 200–201°C. Yield
–CH N); 9.79 (1H, s, –NH). Anal. calcd. for C22H19Cl2N3O2: C,
–
–
61.69; H, 4.47; N, 9.81%. Found: C, 60.84; H, 4.44; N, 9.83%.
83%. Rf ꢁ 100 value: 30 (M2). HPLC tR (min): 3.705. FT-IR (vmax
,
N0-[(4-Bromophenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4h)
Cream-colored solid. MW: 461.351. m.p. 206–207°C. Yield
1
cmꢀ1): 3227 (hydrazone NH), 1682 (C O), 1603 (C N). H NMR
–
–
–
–
(300 MHz, CDCl3): d (ppm) 1.7 (s, –CH3 peak of ethanol held by
the molecule); 2.44 (3H, s, Ar–CH3); 3.81 (s, –CH2 peak of
ethanol that held by the molecule); 3.97 (–OH peak of ethanol
that held by the molecule); 4.04 (3H, s, N–CH3); 4.21 (2H, s,
–CH2–); 6.19 (1H, d, pyrrole ring protons adjacent CH2,
70%. Rf ꢁ 100 value: 35 (M2). HPLC tR (min): 4.489. FT-IR (vmax
,
1
cmꢀ1): 3152 (hydrazone NH), 1686 (C O), 1597 (C N). H NMR
–
–
–
–
(300 MHz, CDCl3): d (ppm) 1.69 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.81 (s, –CH2 peak of
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
S¸. G. Kuc¸ukguzel et al.
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ethanol that held by the molecule); 3.97 (s, –OH peak of
ethanol that held by the molecule); 4.04 (3H, s, N–CH3); 4.21
(2H, s, –CH2–); 6.18 (1H, d, pyrrole ring protons adjacent CH2,
(300 MHz, CDCl3): d (ppm) 1.66 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.81 (s, –CH2 peak of
ethanol that held by the molecule); 3.97 (s, –OH peak of
ethanol that held by the molecule); 4.00 (3H, s, N–CH3); 4.16
(2H, s, –CH2–); 6.14 (1H, d, pyrrole ring protons adjacent CH2,
–
J ¼ 4.06 Hz); 6.71 (1H, d, pyrrole ring protons adjacent C O,
–
–
J ¼ 4.05 Hz); 7.24–7.74 (8H, m, Ar–H); 7.80 (1H, s, –CH N); 10.08
–
–
(1H, s, –NH). Anal. calcd. for C22H20BrN3O2.1/2C2H5OH: C,
J ¼ 4.07 Hz); 6.68 (1H, d, pyrrole ring protons adjacent C O,
–
–
60.28; H, 4.60; N, 9.59%. Found: C, 59.54; H, 4.57; N, 9.62%.
J ¼ 4.06 Hz); 7.07–7.74 (13H, m, Ar–H); 7.79 (1H, s, –CH N); 9.77
–
(1H, s, –NH). Anal. calcd. for C28H25N3O3.1/2C2H5OH: C, 73.4;
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N0-[(4-
nitrophenyl)methylidene]acetohydrazide (4i)
H, 5.95; N, 8.85%. Found: C, 73.76; H, 5.51; N, 9.26%.
Biological methods
Cell treatment
Pale yellow colored solid. MW: 413.427. m.p. 225–228°C. Yield
88%. Rf ꢁ 100 value: 34 (M2). HPLC tR (min): 3.299. FT-IR (vmax
,
1
cmꢀ1): 3180 (hydrazone NH), 1670 (C O), 1599 (C N). H NMR
Human colon carcinoma cell lines HCT-116 (ATCC, CCL-247)
and HT-29 (ATCC, HTB-38), and mouse embryonic fibroblast
cell line NIH3T3 (ATCC, CRL-1658) were maintained in
Dulbecco modified Eagle’s medium (DMEM) supplemented
with 10% FBS and 1% penicillin/streptomycin in the presence
of 5% CO2 in air at 37°C. Tolmetin and compound 4g were
dissolved in DMSO and the cells were treated with increasing
doses of compounds for 24 h in 37°C. After the incubation, the
cells were harvested to analyze apoptosis.
–
–
–
–
(300 MHz, CDCl3): d (ppm) 2.32 (3H, s, Ar–CH3); 3.90 (3H, s,
N–CH3); 4.08 (2H, s, –CH2–); 6.04 (1H, d, pyrrole ring protons
adjacent CH2, J ¼ 4.06 Hz); 6.58 (1H, d, pyrrole ring protons
–
adjacent C O, J ¼ 4.04 Hz); 7.12–7.81; 8.11–8.23 (8H, m, Ar–H);
–
–
7.95 (1H, s, –CH N); 11.43 (1H, s, –NH). Anal. calcd. for
–
C
22H20N4O4.1/2H2O: C, 63.91; H, 5.12; N, 13.55%. Found: C,
64.49; H, 4.95; N, 13.73%.
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N0-
[phenylmethylidene]acetohydrazide (4j)
Cell viability assay
Cream-colored solid. MW: 382.45526. m.p. 168–172°C. Yield
Cell viability was determined by the MTT assay method.
Briefly, HT-29 cells (1 ꢁ 104 cells/well) were seeded into 96-well
plates and incubated for 24 h at 37°C in CO2 incubator. Then,
the cells were grown in the absence or presence of increasing
doses of tolmetin and compound 4g at 37°C. After 24 h
treatment, the media was removed and then 10 mL of MTT
(5 mg/mL in phosphate-buffered saline) was added to each
well for an additional 4 h. The precipitated formazan was
dissolved in 100 mL of 10% SDS and the absorbance was taken
at 570 nm. The percentage of viability was calculated as the
following formula: (viable cells)% ¼ (OD of treated sample/
OD of untreated sample) ꢁ 100.
71%. Rf ꢁ 100 value: 36 (M2). HPLC tR (min): 3.590. FT-IR (vmax
,
1
cmꢀ1): 3194 (hydrazone NH), 1667 (C O), 1605 (C N). H NMR
–
–
–
–
(300 MHz, CDCl3): d (ppm) 1.68 (s, –CH3 peak of ethanol held
by the molecule); 2.44 (3H, s, Ar–CH3); 3.83 (s, –CH2 peak of
ethanol that held by the molecule); 3.98 (s, –OH peak of
ethanol that held by the molecule); 4.05 (3H, s, N–CH3); 4.23
(2H, s, –CH2–); 6.20 (1H, d, pyrrole ring protons adjacent CH2,
–
J ¼ 4.06 Hz); 6.72 (1H, d, pyrrole ring protons adjacent C O,
–
–
J ¼ 4.04 Hz); 7.24–7.75 (9H, m, Ar–H); 7.85 (1H, s, –CH N); 9.82
–
(1H, s, –NH). Anal. calcd. for C22H21N3O2.1/2C2H5OH: C, 72.23;
H, 6.32; N, 10.99%. Found: C, 72.32; H, 5.761; N, 11.65%.
N0-[(4-Methoxyphenyl)methylidene]-2-[1-methyl-5-(4-
methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4k)
Cream-colored solid. MW: 412.481. m.p. 189°C. Yield 84%.
Annexin V binding
After treatment, the cells were pre-incubated on ice for
30 min. Then, cells were centrifuged and resuspended in cold
PBS. Following incubation with annexin V for 30 min, the cells
were subsequently washed and fixed in PFA. The fluorescence
intensity was measured using a fluorospectrophotometer.
Rf ꢁ 100 value: 31 (M2). HPLC tR (min): 3.460. FT-IR (vmax, cmꢀ1):
1
–
–
–
3186(hydrazoneNH), 1668(C O),1622(C N). HNMR(300 MHz,
–
CDCl3): d (ppm) 1.71 (s, –CH3 peak of ethanol held by the
molecule);2.44 (3H, s, Ar–CH3); 3.81 (s, –CH2 peak of ethanol that
held by the molecule); 3.84 (3H, s, N–CH3); 3.98 (s, –OH peak of
ethanol that held by the molecule); 4.05 (3H, s, –OCH3); 4.21
(2H, s, –CH2–); 6.20 (1H, d, pyrrole ring protons adjacent CH2,
Measurement of caspase-3 activity
To quantify caspase-3 activity, a caspase colorimetric assay kit
(Caspase-3 Cellular Activity Assay Kit, Millipore) was used.
Firstly, tissue samples were sonicated in iced lysis buffer for
10 min. Caspase-3 activity was measured spectrophotometri-
cally using tetrapeptide substrate N-acetyl-Asp-Glu-Val-Asp-
p-nitroanilide (AC-DEVD-pNa) in the cytosolic samples. Briefly,
the recommended amount of sample was added into each
well and treated with assay buffer containing 400 mM
substrate (DEVD-pNA) [100 mmol/L NaCl, 50 mmol/L HEPES,
10 mmol/L dithiothreitol, 1 mmol/L EDTA, 10% glycerol, 0.1%
of 3-((3-cholamidopropyl)dimethylammonio)-1-propanesul-
fonic acid, pH 7.4]; then the microplate was equilibrated at
–
J ¼ 4.06 Hz); 6.71 (1H, d, pyrrole ring protons adjacent C O,
–
–
J ¼ 4.05 Hz); 6.89–7.75 (8H, m, Ar–H); 7.80 (1H, s, –CH N); 9.84
–
(1H, s, –NH). Anal. calcd. for C23H23N3O3.1/2C2H5OH: C, 69.88;
H, 6.35; N, 10.19%. Found: C, 70.67; H, 5.876; N, 10.94%.
2-[1-Methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]-N0-[3-
phenoxyphenyl-methylidene]acetohydrazide (4l)
White-colored solid. MW: 474.550. m.p. 172°C. Yield 62%.
Rf ꢁ 100 value: 67 (M2). HPLC tR (min): 4.958. FT-IR (vmax, cmꢀ1):
3231 (hydrazone NH), 1682 (C O), 1612 (C N). 1H NMR
–
–
–
–
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Arch. Pharm. Chem. Life Sci. 2015, 348, 730–742
Tolmetin Hydrazones Against Colon Cancer
Archiv der Pharmazie
37°C for 10 min. The conversion of substrate into the colored
product was measured and recorded at 30-min intervals for
2 h at 405 nm with a microplate reader [40].
were optimized with semiempirical PM6 method [43] via
conformation search in Spartan 10 program [44]. For each
compound, the most stable conformation was chosen
and used for initial geometry in docking calculation. The
AutoDock Tools program [45] was utilized to generate the
docking input files as described before [46]. A grid box size of
40 ꢁ 40 ꢁ 40 points in x, y, and z dimensions was built. For
COX-1 target, the grid was centered at 26.108 (x), 35.061 (y),
and 197.448 (z). Regarding COX-2, the grid was centered at
ꢀ39.746 (x), ꢀ50.781 (y), and ꢀ22.645 (z). A grid spacing of
Measurement of caspases 8 and 9 activity
To measure caspase
8 and caspase 9 activity, caspase
colorimetric assay kits (Millipore, Billerica, MA) were used.
Therefore, cell pellets were lysed in the lysis buffer, and
supernatants were collected. The approximate amount of
samples were incubated with reaction buffer and colorimetric
substrate, acetyl (Ac)-Ile-Glu-Thr-Asp (IETD)-p-nitroaniline
(pNA) for caspase-8 and Ac-Leu-Glu-His-Asp (LEHD)-pNA for
caspase-9, respectively, at 37°C for 2 h in the dark. The
reactions were assessed for changes in absorbance at 405 nm
using a microplate reader. Results are expressed as fold
increase of the activity measured in samples. Fold-increase
activity can be determined by comparing the results of treated
samples with the level of the uninduced control [41].
̊
0.375 A was used for the calculation of the energetic map.
Ligands were chosen fully flexible. The Vina parameter
“exhaustiveness” was set to the value of 10. The charge of
the heme Fe atom was set to þ2 manually. The resultant
structure files were analyzed using Accelrys Discovery Studio
Visualizer 4.1 program [47]. In order to validate the docking
protocol, the co-crystallized inhibitors a-methyl-4-biphenyl-
acetic acid and naproxen were docked into their respective
enzymes COX-1 and COX-2. The docking poses were alligned
to their binding orientation in the pdb files. Their docked
poses were in perfect agreement with pdb poses, validating
our procedure.
Determination of the mitochondrial membrane potential
changes using fluorescent probe JC-1
JC-1, a lipophilic cationic dye, is widely used to detect
mitochondrial depolarization. In healthy cells, the dye stains
the mitochondrial membrane bright red whereas apoptotic
cells were stained green. Briefly, cells were cultured on cover
slips and exposed to drugs for 24 h. After discarding the
culture medium, 1 mL of the JC-1 staining solution was added
to the wells. The cells were then incubated at 37°C for
15 min in a CO2 incubator and washed twice with assay buffer.
A drop of assay buffer was then added to the wells prior
to immediate examination in the fluorescence microscope
(Olympus, Tokyo, Japan).
This work was generously supported by the Research Fund of
Marmara University, project number: SAG-C-YLP-161111-
€
0297. The authors are grateful to Dr. Jurgen 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. Tolmetin was supplied by Santa-
Farma Pharmaceutical Industry Inc. S. S. Erdem thanks
Prof. M. T. Sac¸an for the use of Spartan software.
The authors have declared no conflicts of interest. The authors
alone are responsible for the content and writing of this
article.
COX activity determination
COX activity of the samples was determined using COX
activity assay kit (Cayman Chemicals) according to the kit’s
manual. Inhibitors were added in 100 mM concentration to
the cultures and incubated for 24 h. Cells (>5 ꢁ 105) were
scraped and lysed with sonication. Reactivity was measured by
detecting absorbance at 590 nm using a plate reader (Synergy
H1, Biotek).
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