Synthesis, anticancer activity, and molecular modeling of etodolac-thioether derivatives as potent methionine aminopeptidase (type II) inhibitors

Article abstract of DOI:10.1002/ardp.201700195

A series of (R,S)-1-{[5-(substituted)sulfanyl-4-substituted-4H-1,2,4-triazole-3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indoles (5a–v) were designed and synthesized using a five-step synthetic protocol that involves substituted benzyl chlorides and (R,S)-5-[(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)methyl]-4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones in the final step. The synthesized derivatives were evaluated for cytotoxicity and anticancer activity in vitro using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method against VERO, HEPG2 (human hepatocellular liver carcinoma), SKOV3 (ovarian carcinoma), MCF7 (human breast adenocarcinoma), PC3 and DU145 (prostate carcinoma) cells at 10^?5 M (10 μM) for 24 h. Compounds 5d and 5h showed the best biological potency against the SKOV3 cancer cell line (IC₅₀ = 7.22 and 5.10 μM, respectively) and did not display cytotoxicity toward VERO cells compared to etodolac. Compounds 5k, 5s, and 5v showed the most potent biological activity against the PC3 cancer cell line (IC₅₀ = 8.18, 3.10, and 4.00 μM, respectively) and did not display cytotoxicity. Moreover, these compounds were evaluated for caspase-3, -9, and -8 protein expression and activation in the apoptosis pathway for 6, 12, and 24 h, which play a key role in the treatment of cancer. In this study, we also investigated the apoptotic mechanism and molecular modeling of compounds 5k and 5v on the methionine aminopeptidase (type II) enzyme active site in order to get insights into the binding mode and energy.

Full text of DOI:10.1002/ardp.201700195

Received: 7 September 2017
Revised: 4 February 2018
Accepted: 6 February 2018
|
|
DOI: 10.1002/ardp.201700195
FULL PAPER
Synthesis, anticancer activity, and molecular modeling of
etodolac-thioether derivatives as potent methionine
aminopeptidase (type II) inhibitors
Işıl Çoruh1
Özge Çevik2
Kemal Yelekçi3
Teodora Djikic3
|
|
|
|
Ş. Güniz Küçükgüzel1
1 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Marmara
University, İstanbul, Turkey
Abstract
A
series of (R,S)-1-{[5-(substituted)sulfanyl-4-substituted-4H-1,2,4-triazole-3-yl]-
2 Department of Biochemistry, School of
Medicine, Adnan Menderes University, Aydın,
Turkey
methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indoles (5a–v) were designed and
synthesized using a five-step synthetic protocol that involves substituted benzyl
chlorides and (R,S)-5-[(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)methyl]-
4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones in the final step. The synthe-
sizedderivativeswere evaluated forcytotoxicityandanticanceractivityinvitrousingthe
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric
method against VERO, HEPG2 (human hepatocellular liver carcinoma), SKOV3 (ovarian
carcinoma), MCF7 (human breast adenocarcinoma), PC3 and DU145 (prostate
carcinoma) cells at 10−5 M (10 μM) for 24 h. Compounds 5d and 5h showed the best
biological potency against the SKOV3 cancer cell line (IC50 = 7.22 and 5.10 μM,
respectively) and did not display cytotoxicity toward VERO cells compared to etodolac.
Compounds 5k, 5s, and 5v showed the most potent biological activity against the PC3
cancer cell line (IC50 = 8.18, 3.10, and 4.00 μM, respectively) and did not display
cytotoxicity. Moreover, these compounds were evaluated for caspase-3, -9, and -8
protein expression and activation in the apoptosis pathway for 6, 12, and 24 h, which
play a key role in the treatment of cancer. In this study, we also investigated the
apoptotic mechanism and molecular modeling of compounds 5k and 5v on the
methionine aminopeptidase (type II) enzyme active site in order to get insights into the
binding mode and energy.
3 Faculty of Engineering and Natural Sciences,
Department of Bioinformatics and Genetic,
Kadir Has University, İstanbul, Turkey
Correspondence
Prof. Ş. Güniz Küçükgüzel, Faculty of
Pharmacy, Department of Pharmaceutical
Chemistry, Marmara University, Haydarpaşa,
34668 İstanbul, Turkey.
Email: gkucukguzel@marmara.edu.tr
Funding information
Scientific Research Project Commission of
Marmara University, Grant number: SAG-C-
DRP-041213-0451
K E Y W O R D S
anticancer activity, apoptosis, etodolac, molecular docking, thioethers
Abbreviations: AO/EB, acridine orange/ethidium bromide; DMF, dimethylformamide; FT-IR,
Fourier transform infrared; HR-MS, high resolution mass spectrometry; MCF7, human breast
adenocarcinoma cell line; MetAP, methionine aminopeptidase; MTT, (3-(4,5-dimethyl
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method; NMR, nuclear magnetic
resonance; SAR, structure−activity relationship; TLC, thin layer chromatography.
|
1
INTRODUCTION
Cancer is
a diverse group of diseases characterized by the
proliferation and spread of abnormal cells and a major worldwide
problem. Therefore, the discovery and development of new potent
This work was partly presented at the 3rd International BAU Drug Design Congress, 1–3
October, 2015.
|
Arch Pharm Chem Life Sci. 2018;351:e1700195.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
1 of 16
https://doi.org/10.1002/ardp.201700195

2 of 16
ÇORUH ET AL.
|
and selective anticancer drugs are of high importance in modern
research.
of this compound and hydrazine hydrate were refluxed to obtain
methyl 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)-
acetate 1 and 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-
1-yl)acetohydrazide 2. Compound 2 and several isothiocyanates
yield 1-[2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)-
acetyl]-4-alkyl/aryl thiosemicarbazides 3a–c. The thiosemicarba-
zides react with sodium hydroxide solutions (2 N) and after
cyclization reaction 5-[(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]-
indole-1-yl)methyl]-4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-
thiones 4a–c are gained. To a suspension of 4-substituted-1,2,4-
triazole-3-thione in DMF containing anhydrous K2CO3, substituted
benzyl chlorides were added to form the target compounds 1-{[5-
(substituted)sulfanyl)-4-substituted-4H-1,2,4-triazole-3-yl]methyl}-
1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indoles 5a–v. The syn-
thesis of novel series of etodolac-thioethers 5a–v (Table 1) was
performed as outlined in Scheme 1 according to the literature.[12,13]
The etodolac-thioethers 5a–v are original compounds. All synthe-
sized compounds were checked for purity using TLC and were
characterized by their melting points, elemental analysis, 1H NMR,
13C NMR, and HR-MS spectral data.
Methionine aminopeptidase (MetAP) is a cobalt-containing
metalloprotease and protein-processing enzyme responsible for
the removal of initiator methionine residues from polypeptide
chains during protein synthesis. In eukaryotes, there are two
isoforms of MetAP, MetAP1 and MetAP2. MetAP2 plays a critical
role in cell proliferation and tumor growth. Due to the pivotal role
of these enzymes for angiogenesis, MetAP2 has been one of the
major targets in the anticancer drug development area. 1,2,4-
Triazole-3-thiones have wide spectrum biological activity.[1] In
recent years, 1,2,4-triazole derivatives and thioethers are reported
as potential MetAP2 inhibitors.[2,3] JNJ-4929821, which contains
triazole structure and 3-anilino-5-benzylthio-1,2,4-triazole deriv-
atives, was identified as the inhibitors of the human metal-
loprotease (MetAP2).[4,5] Hou and co-workers reported that 1,4-
benzodioxan fragment is an important pharmacophore on biologi-
cal activity, interaction 1,4-benzodioxane and the hydrogen of
some amino in the active site of MetAP2 and this condition
increased biological activity.
Etodolac, (R,S)-2-[1,8-diethyl-1,3,4-tetrahydropyrano[3,4-b]in-
The FT-IR spectral data of etodolac thioethers 5a–v have exhibited
bands in the region 3169–3293 cm−1 which may be attributed to indole
N─H stretching. The N─H bending and C─N stretching bands of the
dole-1-yl]acetic acid, is
a non-steroidal anti-inflammatory drug
which has anticancer activities reported in the literature.[6,7] In
addition, etodolac hydrazones,[8] thiosemicarbazides,[9] 1,2,4-
triazoles,[10] 1,2,3-triazoles,[11] and 4-thiazolidinones[8] have been
reported to have anticancer effect and to be hepatitis C NS5B
polymerase inhibitors. Etodolac have tetrahydropyrano group. There
was no study showing the MetAP2 interaction of the tetrahydropyr-
ano group. In the light of these literature, etodolac was chosen as a
starting substance to synthesize thioether containing 1,2,4-triazole
compounds.

indole structure and the aromatic C C stretching bands of the
compounds, whose practical results are in agreement with literature, are
located at the range of 1421–1622 cm−1. These values are in agreement
with the values reported.[14]
TABLE 1 Structural profiles of compounds 5a–v
Compounds
R1
R2
The aim of this study was to synthesize novel and original 19
thioether compounds starting from etodolac, racemic form, and to
evaluate and confirm their structures by FTIR, 1H NMR, 13C NMR,
high resolution mass spectrometry (HR-MS) spectral data and
elemental analysis data. The synthesized compounds were evaluated
on the several cancer cell lines in the following studies. Our strategy
opens up possibilities for the design and synthesis of new thioether
moieties with anticancer activities.
5a
5b
5c
5d
5e
5f
CH3−
C6H5−CH2−
CH3−
(4)F−C6H4−CH2−
(2)Cl−C6H4−CH2−
(4)Cl−C6H4−CH2−
(2,6)Cl2−C6H3−CH2−
(4)CH3−C6H4−CH2−
(4)NO2−C6H4−CH2−
C6H5−CH2−
CH3−
CH3−
CH3−
CH3−
5g
5h
5i
CH3−
In continuation to extend these research areas on anticancer
compounds with MetAP2 structure inhibitory activity, in the present
work a series of etodolac-thioether derivatives derived from 1,2,4-
triazole-3-thione have been studied. Our study may also allow the
identification of key substrates by database mining.
CH3−CH2−
CH3−CH2−
CH3−CH2−
CH3−CH2−
CH3−CH2−
CH3−CH2−
C6H5−
(4)F−C6H4−CH2−
(2)Cl−C6H4−CH2−
(4)Cl−C6H4−CH2−
(2,6)Cl2−C6H3−CH2−
(4)CH3−C6H4−CH2−
C6H5−CH2−
5j
5k
5l
5m
5n
5p
5r
|
2
RESULTS AND DISCUSSION
C6H5−
(4)F−C6H4−CH2−
(4)Cl−C6H4−CH2−
(2,6)Cl2−C6H3−CH2−
(4)CH3−C6H4−CH2−
(4)NO2−C6H4−CH2−
|
2.1 Chemistry
C6H5−
Etodolac, (R,S)-2-[1,8-diethyl-1,3,4-tetrahydropyrano[3,4-b]indole-
1-yl]acetic acid, was refluxed in the presence of methanol by
catalyst saturated sulfuric acid to form methyl 2-(1,8-diethyl-1,3,4,9-
tetrahydropyrano[3,4-b]indole-1-yl)acetate 1. Methanolic solution
5s
5t
C6H5−
C6H5−
5v
C6H5−

ÇORUH ET AL.
3 of 16
|
SCHEME 1 Synthesis of compounds 5a–v. Reagents and conditions: (i) CH3OH/H2SO4; (ii) NH2NH2.H2O; (iii) R1─NCS (R1: ─CH3,
─CH2CH3, C6H5)/EtOH; (iv) NaOH (2 N), HCl; (v) DMF, K2CO3, R2─CH2─Cl
When 1H NMR spectra of etodolac thioethers 5a–vare considered, the
first spectroscopic evidence of thioether structure formation is the
observation of integral values at approximately 4.00 ppm, which is assigned
to S-CH2 protons.[15–17] Mostly, the S-CH2 protons are determined as
singlets. Depending on the substituent in the compounds 5e, 5g, 5j–l, 5n, 5s,
5t, and 5v, geminal protons in the form of double doublets were detected
and coupling constants were calculated as 12 and 15 Hz.
correlated for the HMBC spectrum evaluation. The 1H and 13C NMR
spectra of the compound are in agreement with the HMBC spectrum at
600 MHz.
The HR-MS results of the compounds 5a, 5c, 5e, 5k, 5n in the
etodolac-thioether structure were detected by electron ionization (EI+)
technique. Molecular weight and elemental composition of the
compounds, m/z values of the resulting mass fractions were determined
by monoisotopic mass values from closed formulas. It has been found
that calculated and found m/z values of the molecular ion peaks of the
compounds, the elementalcompositionandthemonoisotopicmassdata
belonging to them, are compatible with each other.
The indole N─H proton of the etodolac thioethers 5a–v in the
main skeleton of etodolac was determined as a singlet between 9.78
and 10.72 ppm. These values are in agreement with the values of
etodolac starting material whose indole N─H value is observed at
10.57 ppm in the 1H NMR spectrum.
Looking at 13C NMR for the compounds 5a, 5c–e, 5h, 5j, and 5n,
|
2.2 Biological evaluation
chemical shifts of the S-CH2 appeared upfield at 39.16, 37.90, 34.97,

38.79, 38.83, and 37.63 ppm, respectively, C N appeared downfield
|
2.2.1 Anticancer activity and apoptosis of etodolac
at 149.64, 149.84, 149.69, 149.00, 149.98, 149.33, and 151.31 ppm,
respectively, and C─S carbon appeared also downfield at 153.58,
153.43, 153.52, 153.85, 152.65, 152.77, and 153.46 ppm, respec-
tively. The 1H and 13C NMR spectra of the compound 5d were
thioethers
The synthesized 19 compounds were tested for anticancer activity in
six different cell lines; VERO cells (African green monkey kidney cell),

4 of 16
ÇORUH ET AL.
|
FIGURE 1 Cell survival of VERO cells incubated with different concentrations of etodolac and (A–C) derivative compounds 5a–v for 24 h.
(D) Cell survival of different cell lines with incubated 10 μM concentration of etodolac, 5d and 5h compounds for 24 h. (+p < 0.05, +++p < 0.001
compare to control groups in VERO cells; ##p < 0.01, ###p < 0.001 compare to control groups in HEPG2 cells; ***p < 0.001 compare to control
a
groups in SKOV3 cells; &p < 0.05, &&p < 0.01, &&&p < 0.001 compare to control groups in MCF7 cells; p < 0.05, aap < 0.01, compare to control
groups in PC3 cells)
HEPG2 (human hepatocellular liver carcinoma), SKOV3 (ovarian
carcinoma), MCF7 (human breast adenocarcinoma), PC3 and DU145
with etodolac, these compounds do not show cytotoxicity against
VERO cells.
(human prostate carcinoma) cells using MTT assay method at 10−5
M
Apoptosis, known as programmed cell death, involves the
controlled dismantling of the cell by cleaving key cellular
components while damaged or redundant. Caspases are a family
of genes important for maintaining cell death via demolishing key
structural proteins and endoproteases that cysteinyl aspartate
proteinases (cysteine proteases that cleave their substrates
following an Asp residue). Nonetheless, caspase-3 is known as
effector caspases, caspase-9 is responsible for the intrinsic
mitochondrial apoptosis and caspase-8 is initiate of extrinsic death
receptor-mediated apoptosis.
(10 μM).[18–20] Dose-dependent cytotoxicity tests were performed in
VERO cells for etodolac thioethers 5a–v. 1-{[5-(4-Chlorobenzyl)-
sulfanyl-4-methyl-4H-1,2,4-triazol-3-yl]methyl}-1,8-diethyl-1,3,4,9-
tetrahydropyrano[3,4-b]indole 5d and 1-{[5-benzylsulfanyl-4-ethyl-
4H-1,2,4-triazol-3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano
[3,4-b]indole 5h compounds were found to be cytotoxic at 10−5
M
concentration (Figure 1).
1-{[5-(4-Chlorobenzyl)sulfanyl)-4-methyl-4H-1,2,4-triazole-3-yl]-
methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole 5d and 1-
{[5-benzylsulfanyl]-4-ethyl-4H-1,2,4-triazole-1,3,4,9-tetrahydropy-
rano[3,4-b]indole 5h compounds have anticancer activity in SKOV3
cancer cells. While etodolac showed no inhibition against SKOV3
cells. Compounds 5a–v inhibited cell proliferation with IC50 values
between 5.10 and 266.9 μM as shown in Table 2. As a result, 1-{[5-(4-
chlorobenzyl)sulfanyl-4-methyl-4H-1,2,4-triazol-3-yl]methyl}-1,8-di-
ethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole 5d and 1-{[5-benzylsul-
fanyl-4-ethyl-4H-1,2,4-triazole-1,3,4,9-tetrahydropyrano[3,4-b]in-
dole 5h were found to have anticancer activity in SKOV3 cell lines
at an IC50 of 7.22 and 5.10 µM, respectively (Figure 2). Compared
These compounds have also been assessed for caspase 3, 9,
and 8 protein expression and activity at the 6th, 12th, and 24th
hours of apoptosis, which can be said to play a key role in cancer
treatment. Following this study apoptotic caspases protein
expression Western blotting bands in SKOV3 ovarian cancer
cells with incubated 10 μM concentration of etodolac, 5d and 5h
compounds. AO/EB staining was performed in SKOV3 cancer
cells to demonstrate cell death by fluorescence microscopy and
time-dependent cell growth in SKOV3 ovarian cancer was
investigated (Figure 3).

ÇORUH ET AL.
5 of 16
|
|
TABLE 2 IC50 values of compounds 5a–v on SKOV3 cancer cell lines
2.3 Molecular modeling
Compound
IC50 (μM)
9.44
Compounds were docked into human MetAP2 (hMetAP2) accord-
ing to the method described in Ref. [21]. The results showed that all
of these compounds have good binding affinity for MetAP2
(Table 4).
Etodolac
5a
110.2
16.00
31.00
7.22
5b
5c
Most important amino acids of the binding pocket that were
making non-bonded hydrophobic interactions with compounds were
HIS231, LEU328, ASN329, HIS331, HIS339, THR343, ASP376,
TYR444, PRO443, and LEU447. Additionally, compounds with
─NO2 group in para position of the phenyl ring made hydrogen
bonds with ARG337 and PHE387. According to docking results, the
compound 5k with ΔG = −11.21 kcal/mol and Ki = 6.10 nM, com-
pound 5s with ΔG = −11.02 kcal/mol and Ki = 8.36 nM, and 5v
11.02 kcal/mol and Ki = 7.27 nM were shown to be the best
inhibitors.
5d
5e
5f
57.2
89.2
5g
5h
5i
246.20
5.10
83.8
5j
113.6
6.48
5k
5l
Furthermore from these results we may conclude that the nitro
group in para position (5v) for compounds containing phenyl in
position 4 of 1,2,4-triazole and dichloro groups at 2,6-positions
(5k) for compounds containing ethyl group at position 4 of 1,2,4-
triazole ring turned out to be important for binding. These
compounds showed good binding for the MetAP2 protein
(Figures 6 and 7). In the same time, they were also most active
in PC3 cancer cell line.
40.3
5m
5n
5p
5r
266.9
19.77
27.5
216.49
9.03
5s
5t
47.575
11.08
5v
|
3
CONCLUSION
Etodolac thioethers 5a–v also were studied on the dose-
dependent cytotoxicity effect of PC3 (androgen-independent
prostate cancer) cells. This study was examined by time-dependent
cell growth on PC-3 prostate cancer cell lines. These compounds
have also been assessed for caspase 3 activity at the 12th, 24th, and
48th hours of apoptosis (Figures 4 and 5).
The selective COX-2 inhibitor etodolac, the carboxylic acid derivative
containing the pyrano[3,4-b]indole structure, has been a non-steroidal
agent with analgesic, antipyretic, and anti-inflammatory effects since
1985. The indole structure is important in the development of new
drug candidates, especially in terms of anticancer activity. Indole-
based pharmacophores are the most preferred structures among
researchers in structural modifications, as indole derivatives respond
to anticancer agents in a significant way. The main reason for selecting
etodolac as the starting material in the synthesis is its anticancer
activity.
While etodolac showed no inhibition against PC3 cells, com-
pounds 5a–v inhibited cell proliferation with IC50 values between 3.10
and 575.93 μM as shown in Table 3. As a result, 1-{[5-(4-chlorobenzyl)-
sulfanyl-4-ethyl-4H-1,2,4-triazole-3-yl]methyl}-1,8-diethyl-tetrahydro-
pyrano[3,4-b]indole 5k, 1-{[5-(2,6-dichlorobenzyl)sulfanyl-4-phenyl-
4H-1,2,4-triazol-3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indole 5s, and 1-{[5-(4-nitrobenzyl)sulfanyl-4-phenyl-1,2,4-
triazol-3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
5v showed the most anticancer activity at 8.18 µM, 3.10, and 4.00 µM
IC50 levels in PC3 cancer cell lines.
Etodolac thioethers were synthesized with substituted benzyl
chlorides and their corresponding etodolac 1,2,4-triazoles. The
cytotoxicity and anticancer activity of synthesized compounds
were evaluated in vitro using the MTT colorimetric method
against VERO cells, HEPG2 (human hepatocellular liver carci-
noma), SKOV3 (ovarian carcinoma), MCF7 (human breast
adenocarcinoma), PC3 and DU145 (prostate carcinoma) cells at
10−5 M for 24 h. Spectroscopic and elemental analysis methods of
the synthesized etodolac thioether derivatives have been
elucidated.
The structure–activity relationship (SAR) analysis stated that
compounds containing electron-withdrawing group in benzyl group
showed stronger anticancer activity against SKOV3 and PC3 cells. The
electron-withdrawing group containing NO2 (5v) and Cl (5s) groups in
benzyl group for phenyl containing at 4-position of 1,2,4-triazole and
Cl (5k) groups in benzyl group for ethyl containing at 4-position of
1,2,4-triazole in prostate cancer cell line. The electron-withdrawing
group containing phenyl (5h) in benzyl group for ethyl containing at 4-
position of 1,2,4-triazole and Cl (5d) groups in benzyl group for methyl
containing at 4-position of 1,2,4-triazole in ovarian cancer cell line.
In this study, anticancer activities of etodolac derivatives
were screened in cell lines VERO (monkey kidney cancer), HEPG2
(human liver cancer), SKOV3 (human ovarian cancer), MCF7
(human breast cancer), PC3 and DU145 (human prostate cancer).
Among these compounds, compounds 5d and 5h were found to
have anticancer activity in SKOV3 cell line at IC50 values of 7.22

6 of 16
ÇORUH ET AL.
|
FIGURE 2 (A) Cell growing imaging and (B) percentage of cell growing SKOV3 ovarian cancer cells with incubated 10 μM concentration of
etodolac, 5d and 5h compounds for 6, 12, and 24 h. (##p < 0.01 compare to control groups in 6 h; ++p < 0.01, +++p < 0.001 compare to control
groups in 12 h, ***p < 0.001 compare to control groups in 24 h. (C) AO/EB staining image and (D) ratio of AO/EB staining in SKOV3 ovarian
cancer cells with incubated 10 μM concentration of etodolac, 5d and 5h compounds for 24 h (***p < 0.001 compare to control groups)
and 5.10 µM, respectively, and IC50 values of 5k, 5s, and 5v
compounds at PC3 cell line at 8.18 µM, 3.10 and 4.00 µM,
respectively.
(TLC) plates precoated with silica gel F254 using solvent systems
M1, petroleum ether/acetone (60:40, v/v); M2, petroleum ether/
acetone (50:50, v/v); M3, petroleum ether/acetone (70:30, v/v).
The spots were located under UV light (254 nm, t = 25°C).
Elemental analyses of compounds were performed on a VarioMI-
CRO V1.5.7. instrument. Infrared spectra of compounds were
recorded on Shimadzu FTIR-8400S spectrophotometer and ex-
pressed in wavenumber ν (cm−1). 1H NMR spectra were recorded
as solutions in CDCl3 and DMSO-d6 on Bruker Ultrashield TM and
Varian Mercury spectrometer operating at 300 and 600 MHz.
13C NMR spectra were recorded on Bruker Avance DPX 400 as
solutions in CDCl3 operating at 75 and 150 MHz. The 1H and
13C chemical shifts were expressed in ppm relative to tetrame-
thylsilan (TMS) as an internal standard, where TMS (δ) = 0.0 ppm.
The multiplicity of the signal is indicated as: d, doublet; m,
multiplet; s, singlet; t, triplet; q, quarted. Coupling constants are
expressed in hertz (Hz). All NMR spectra and elemental analyses
were carried out by the Inonu University Scientific and Techno-
logical Research Center (IBTAM).
A molecular modeling study using MetAP2 enzyme to support
these findings were performed and compound 5k with the values of
ΔG = −11.21 kcal/mol, Ki = 6.10 nM, 5s with the values of
ΔG = −11.02 kcal/mol, Ki = 8.36 nM, and 5v with the values of
ΔG = −11.02 kcal/mol, Ki = 7.27 nM were found to be the best MetAP2
inhibitors. With these results, there was a correlation between
molecular modeling and anticancer activity results.
|
4
EXPERIMENTAL
|
4.1 Chemistry
|
4.1.1 General
All solvents and chemicals used in this study were supplied from
Sigma–Aldrich, Merck and used without purification. Melting
points (mp) were determined using Schmelzpunktbestimmer SMP
II melting point apparatus and expressed in degrees centigrade (°C).
Reactions were monitored by analytical thin layer chromatography
The IR and NMR spectra as well as the InChI codes of the
investigated compounds together with some biological activity data
are provided as Supporting Information.

ÇORUH ET AL.
7 of 16
|
FIGURE 3 (A) Apoptotic caspases protein expression Western blotting bands in SKOV3 ovarian cancer cells with incubated 10 μM
concentration of etodolac, 5d and 5h compounds for 6, 12, and 24 h. (B) Densitometry analysis of Cas3/β-actin and Clv-Cas3/β-actin protein
expression levels (***p < 0.001 compare to Cas3 bands of control groups; +++p < 0.001 compare to Clv-Cas3 bands of control groups). (C)
++
Densitometry analysis of Cas8/β-actin and Clv-Cas8/β-actin protein expression levels (**p < 0.01 compare to Cas8 bands of control groups;
+p < 0.001 compare to Clv-Cas8 bands of control groups). (D) Densitometry analysis of Cas9/β-actin protein expression levels (***p < 0.001
compare to Cas9 bands of control groups). (E) Caspase3 activity in SKOV3 ovarian cancer cells with incubated 10 μM concentration of 5d and
5h compounds for 6, 12, and 24 h (*p < 0.05, ***p < 0.001 compare to control groups of each compound)
|
4.1.2 Preparation of (R,S)-methyl-2-(1,8-diethyl-
1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)acetate
(1, CAS no. 122188-02-07) and (R,S)-2-(1,8-diethyl-
1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)-
acetohydrazide (2, CAS no. 946848-58-4)
reflux for 2 h. The precipitate obtained was filtered off,
washed with water, and followed by two washings with boiling
ethanol.[9]
|
4.1.4 General procedure for synthesis of (R,S)-5-[(1,8-
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 sodium
bicarbonate (NaHCO3) (5%). The resulting precipitate was filtered,
dried, and recrystallized twice from ethanol. Methanolic solution of
etodolac ester (0.01 mol) and hydrazine hydrate (80%, 7 mL) were
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. (For compound 2 mp 186–188°C[8].)
diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-yl)-
methyl]-4-substituted-2,4-dihydro-3H-1,2,4-triazole-
3-thiones (4a–c; 4a CAS no. 1526911-68-1, 4b CAS
no. 1528513-78-1, 4c CAS no. 1528513-91-8)
A sample of etodolac thiosemicarbazides 3a–c (0.001 mol) and sodium
hydroxide (NaOH) solution (2 N) was refluxed for 4 h on water bath.
On cooling, it was solidified. This was dissolved in water and acidified
with hydrochloric acid (HCl) solution (10%). The precipitate was
washed with water and recrystallized from ethanol.[10]
|
|
4.1.5 General procedure for synthesis of (R,S)-1-{[5-
4.1.3 General procedure for the synthesis of (R,S)-
(substituted)sulfanyl-4-substituted-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano-
[3,4-b]indole (5a–v)
1-[2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]-
indole-1-yl)acetyl]-4-alkyl/arylthiosemicarbazides
(3a–c; 3a CAS no. 1526911-69-2, 3b CAS no.
1528514-00-2, 3c CAS no. 1528514-12-6)
To a suspension of substituted-1,2,4-triazole-3-thione 4a–c (0.7 g,
1.9 mmol) in N,N-dimethylformamide (DMF) (5 mL) containing anhy-
drous potassium carbonate (K2CO3) (0.26 g, 1.9 mmol), substituted
A solution of compound 2 (0.01 mol) and equimolar amount of
appropriate isothiocyanate in 20 mL of ethanol was heated under

8 of 16
ÇORUH ET AL.
|
FIGURE 4 Cell survival of PC3 cells incubated with different concentrations of etodolac and (A–C) derivative compounds 5a–v for 24 h
benzyl chloride (1.9 mmol) was added. The reaction mixture was stirred
at room temperature for overnight, then poured onto cold water and
the formed precipitate was filtered, washed with water, dried, and
crystallized.
198.0920 (2.34), 156.0813 (2.71), 91.0548 (11.86), 57.0339 (4.00).
Analysis for C26H30N4OS.1/2H2O; calcd. (%): C, 68.54; H, 6.86; N,
12.30; S, 7.04. Found: C, 68.78; H, 6.61; N, 12.34; S, 6.81.
(R,S)-1-{[5-(4-Fluorobenzyl)sulfanyl-4-methyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5b)
(R,S)-1-{[5-Benzylsulfanyl-4-methyl-4H-1,2,4-triazol-3-yl]-
methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (5a)
White solid, yield 62.55%, mp 162.0–163.0°C. Rf: 0.37 (M1). FT-IR
(vmax, cm−1): 3182 (indole N─H); 3084, 3028, 2964, 2928, 2860 (C─H);
``Pale yellow solid, yield 47.30%, mp 161.1°C. Rf: 0.10 (M1). FT-IR
(vmax, cm−1): 3254 (indole NH), 3017, 2963, 2920, 2872 (C─H);




1504, 1496, 1471, 1458, 1440 ((N─H, C C, triazole C N); 1369,
1329 (C─H); 1249 (C─S); 1076 (pyran C─O); 801, 754 (monosub-
stituted benzene); 696 (C─S). 1H NMR (600 MHz, DMSO-d6): δ 0.58 (t,
3H, ─CH2─CH3 at C1, J = 7.2 Hz, J = 7.8 Hz); 1.27 (t, 3H, ─CH2─CH3 at
C8, J = 7.2 Hz, J = 7.8 Hz); 1.56–2.07 (m, 2H, ─CH2─CH3 at C1); 2.50–
2.89 (m, 4H, ─CH2─CH3 at C8, ─CH2 at C1); 3.31–3.44 (m, 5H, ─CH2
at C4 and N─CH3); 3.78–3.90 (m, 2H, ─CH2 at C3); 4.26 (s, 2H, S─CH2);
6.89–7.29 (m, 8H, Ar─H); 10.70 (s, 1H, indole N─H). 13C NMR
(150 MHz, CDCl3): δ 7.61 (C-12); 13.79 (C-10); 22.49 (N-CH3); 24.29
(C-9); 30.05 (C-4); 30.45 (C-11); 34.81 (C-13); 36.83 (S-CH2); 60.98
(C-3); 76.24 (C-1); 108.08 (C-4a); 115.77 (C-6); 119.45 (C-5); 120.27
(C-7); 126.01 (C-5a); 126.98, 127.08 (C-2′ and C-6′); 129.38 (C-4′);
129.80, 131.00 (C-3′ and C-5′); 134.17 (C-8); 134.64 (C-1′); 134.68
1601, 1508, 1472, 1460, 1413 (N─H, C C, triazole C N); 1375,
1292 (C─H); 1225 (C─S); 1080 (pyran C─O); 742, 696 (benzene);
657 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.77 (t, 3H, ─CH2─CH3 at
C1, J = 7.2 Hz); 1.37 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz, J = 7.8 Hz);
1.98–2.3 (m, 2H, ─CH2─CH3 at C1); 2.80–2.93 (m, 4H, ─CH2─CH3
at C8 and ─CH2─ at C1); 3.09–3.25 (m, 5H, ─CH2─ at C4 and
─N─CH3); 3.98–4.13 (m, 2H, ─CH2 at C3); 4.31 (s, 2H, S─CH2);
6.95–7.36 (m, 7H, Ar─H); 10.04 (s, 1H, indole N─H). 13C NMR
(75 MHz, CDCl3): δ 7.58 (C-12); 13.78 (C-10); 22.47 (N─CH3); 24.26
(C-9); 30.14 (C-4); 30.56 (C-11); 34.73 (C-13); 37.92 (S─CH2); 60.96
(C-3); 76.30 (C-1); 108.19 (C-4a); 115.48 (C-6); 119.48 (C-5); 120.29
(C-7); 126.03 (C-5a); 127.04, 130.58 (C-2′ and C-6′); 130.69 (C-4′);
132.61, 132.65 (C-3′ and C-5′); 134.66 (C-8); 135.70 (C-1′); 149.83


(C-1a); 135.78 (C-8a); 149.64 (triazole C N); 153.58 (C─S). HR MS
[(EI+), m/z (%)]: 446.2143 ([M+], 9.16), 417.1760 (2.33), 229.1422
(C-1a); 153.46 (C-8a); 160.66 (triazole C N); 163.94 (C─S). Analysis
for C26H29FN4OS calcd. (%): C, 67.21; H, 6.29; N, 12.06; S, 6.90.
(17.54), 228.1390 (100.0), 227.1308 (15.58), 219.0832 (5.81),
Found (%): C, 66.55; H, 6.68; N, 11.36; S, 6.35.

ÇORUH ET AL.
9 of 16
|
FIGURE 5 (A) Percentage of cell growing PC3 prostate cancer cells incubated with 10 μM concentration of etodolac, 5k, 5s, and 5v
compounds for 12, 24, and 48 h (##p < 0.01 compare to control groups in 12 h; ++p < 0.01, +++p < 0.001 compare to control groups in 24 h,
***p < 0.001 compare to control groups in 48 h). (B) Cell survival of different prostate cancer PC3 and DU145 cell lines incubated with 10 μM
concentration of etodolac, 5k, 5s, and 5v compounds for 36 h (+p < 0.05, ++p < 0.01 compare to control groups in PC3 cells; #p < 0.05,
##p < 0.01, ###p < 0.001 compare to control groups in DU145 cells). (C) Caspase3 activity in PC3 prostate cancer cells incubated with 10 μM
concentration of 5k, 5s, and 5v compounds for 12, 24, and 48 h (*p < 0.05, ***p < 0.001 compare to control groups of each compound)
(R,S)-1-{[5-(2-Chlorobenzyl)sulfanyl-4-methyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5c)
(3.19), 198.0914 (2.03), 156.0810 (2.20), 125.0157 (5.60), 57.0338
(4.29). Analysis for C26H29ClN4OS.H2O calcd. (%): C, 62.57; H, 6.26; N,
11.23; S, 6.42. Found (%): C, 61.78; H, 6.15; N, 11.30; S, 6.32.
White solid, yield 52.0%, mp 153.4°C. Rf: 0.19 (M1). FT-IR (vmax, cm−1):
3526 (O─H); 3246 (indole N─H); 3061, 2970, 2918, 2862 (C─H);
(R,S)-1-{[5-(4-Chlorobenzyl)sulfanyl-4-methyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5d)


1601, 1582, 1520, 1476, 1447, 1422 (N─H, C C, triazole C N);
1329, 1319 (C─H); 1252 (C─S); 1072 (pyran C─O); 743, 692 (o-
substituted benzene); 677 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.76 (t,
3H, ─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.37 (t, 3H, ─CH2─CH3 at
C8, J = 7.5 Hz, J = 7.8 Hz); 1.97–2.29 (m, 2H, ─CH2─CH3 at C1); 2.79–
2.94 (m, 4H, ─CH2 ─CH3 at C8 and ─CH2─ at C1); 3.07–3.24 (m, 5H,
─CH2─ at C4 and ─N─CH3); 3.98–4.13 (m, 2H, ─CH2 at C3); 4.41 (s,
2H, S─CH2); 7.01–7.38 (m, 7H, Ar─H); 10.13 (s, 1H, indole N─H).
13C NMR (75 MHz, CDCl3): δ 7.57 (C-12); 13.75 (C-10); 22.47
(N─CH3); 24.24 (C-9); 30.03 (C-4); 30.45 (C-11); 34.76 (C-13); 39.16
(S─CH2); 60.95 (C-3); 76.26 (C-1); 108.05 (C-4a); 115.73 (C-6); 119.42
(C-5); 120.22 (C-7); 126.01 (C-5a); 127.08 (C-2′ and C-6′); 127.81 (C-
4′); 128.70 (C-3′ and C-5′); 128.89 (C-8); 134.67 (C-1′); 135.80 (C-1a);
136.80 (C-8a); 149.84 (triazole CN); 153.43 (C─S). HR-MS [(EI+), m/z
(%)]: 482.1728 ([M+2]+, 2.78), 480.1747 ([M+], 6.80), 253.0437 (4.47),
229.1422 (16.77), 228.1383 (100.0), 227.1305 (15.49), 226.1231
White solid, yield 38.4%, mp 116.0–119.0°C. Rf: 0.53 (M1). FT-IR
(vmax, cm−1): 3600 (O─H); 3169 (indole N─H); 3082, 2963, 2920,

2872, 2861 (C─H); 1592, 1503, 1489, 1470, 1449 (N─H, C C,

triazole C N); 1375, 1314 (C─H); 1246 (C─S); 1080 (pyran C─O);
750, 669 (p-substituted benzene); 628 (C─S). 1H NMR (300 MHz,
CDCl3): δ 0.77 (t, 3H, ─CH2─CH3 at C1, J = 7.5 Hz, J = 7.2 Hz); 1.36 (t,
3H, ─CH2─CH3 at C8, J = 7.5 Hz, J = 7.8 Hz); 1.98–2.30 (m, 2H,
─CH2─CH3 at C1); 2.76–2.92 (m, 4H, ─CH2─CH3 at C8 and ─CH2─ at
C1); 3.14–3.25 (m, 5H, ─CH2─ at C4 and ─N─CH3); 3.98–4.13 (m, 2H,
─CH2 at C3); 4.30 (s, 2H, S─CH2); 6.99–7.37 (m, 7H, Ar─H); 10.0317
(s, 1H, indole N─H). 13C NMR (75 MHz, CDCl3): δ 7.62 (C-12); 13.81
(C-10); 22.48 (N─CH3); 24.27 (C-9); 30.18 (C-4); 30.62 (C-11); 34.72
(C-13); 37.90 (S─CH2); 60.97 (C-3); 76.32 (C-1); 108.24 (C-4a); 115.81
(C-6); 119.50 (C-5); 120.30 (C-7); 126.05 (C-5a); 127.05 (C-2′ and C-

10 of 16
ÇORUH ET AL.
|
TABLE 3 IC50 values of 5a–v on PC3 prostate cancer cell line
6′); 128.86 (C-4′); 130.31 (C-3′ and C-5′); 133.72 (C-8); 134.70 (C-1′);

135.41 (C-1a); 135.69 (C-8a); 149.69 (triazole C N); 153.52 (C─S).
Compound
IC50 (μM)
20.29
40.75
77.97
208.12
11.20
234.43
48.83
575.93
15.01
75.06
88.79
8.18
Analysis for C26H29ClN4OS.3/2 H2O calcd. (%): C, 61.46; H, 6.35; N,
Etodolac
5a
11.03; S, 6.31. Found (%): C, 61.43; H, 5.97; N, 10.32; S, 5.81.
5b
5c
(R,S)-1-{[5-(2,6-Dichlorobenzyl)sulfanyl-4-methyl-4H-1,2,4-
triazole-3-yl]methyl}-1,8-diethyl-tetrahydropyrano[3,4-b]indole
(5e)
5d
5e
5f
White solid, yield 68.0%, mp 145.0–147.0°C. Rf: 0.57 (M2). FT-IR
(vmax, cm−1): 3293 (indole N─H); 3053, 2964, 2926, 2868 (C─H); 1558,


1501, 1468, 1434 (N─H, C N, C C); 1374, 1335 (C─H); 1250 (C─S);
1075 (pyran C─O); 783, 741 (substituted benzene); 693 (C─S).
1H NMR (300 MHz, CDCl3): δ 0.78 (t, 3H, ─CH2─CH3 at C1, J = 7.2 Hz,
J = 7.5 Hz); 1.35 (t, 3H, ─CH2─CH3 at C8, J = 7.8 Hz, J = 7.5 Hz); 1.98–
2.26 (m, 2H, ─CH2─CH3 at C1); 2.81–2.92 (m, 4H, ─CH2 ─CH3 at C8
and ─CH2─ at C1); 3.11–3.28 (m, 5H, ─CH2─ at C4 and ─N─CH3);
4.01–4.12 (m, 2H, ─CH2 at C3); 4.45–4.59 (dd, 2H, J = 12.6 Hz,
J = 12.9 Hz, S─CH2); 7.0–7.36 (m, 6H, Ar─H); 10.33 (s, 1H, indole
N─H). 13C NMR (75 MHz, CDCl3): δ 7.64 (C-12); 13.76 (C-10); 22.51
(N─CH3); 24.26 (C-9); 30.28 (C-4); 34.91 (C-11, C-13); 34.97 (S─CH2);
60.99 (C-3); 76.29 (C-1); 107.93 (C-4a); 115.73 (C-6); 119.41 (C-5);
120.19 (C-7); 126.01 (C-5a); 127.17 (C-2′ and C-6′); 128.50 (C-4′);
5g
5h
5i
5j
5k
5l
ND
5m
5n
5p
5r
ND
62.12
80.53
15.05
3.10
5s
129.52 (C-3′ and C-5′); 133.00 (C-8); 134.69 (C-1′); 135.75 (C-1a);
5t
28.24
4.00
+

135.93 (C-8a); 149.00 (triazole C N); 153.85 (C─S). HR-MS [(EI ), m/z
5v
(%)]: 518.1311 ([M+4]+, 0.85), 516.1322 ([M+2]+, 3.84), 514.1348
([M+], 5.29), 287.0041 (4.13), 229.1415 (16.80), 228.1377 (100.0),
227.1299 (15.15), 160.9733 (4.59), 158.9763 (7.15), 156.0806 (2.17),
57.0338 (4.08). Analysis for C26H28Cl2N4OS.1/2H2O calcd. (%): C,
59.54; H, 5.57; N, 10.68; S, 6.11. Found (%): C, 59.01; H, 5.19; N, 10.52;
S, 6.47.
ND, not determined.
TABLE 4 Docking scores of compounds 5a–v
AutoDock 4.2.6
Compounds
ΔG (kcal/mol)
−9.20
Ki (nM)
179.32
345.21
227.02
104.89
55.30
129.97
639.86
227.39
216.94
150.68
6.10
5a
5b
5c
5d
5e
5f
(R,S)-1-{[5-(4-Methylbenzyl)sulfanyl-4-methyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5f)
−8.82
−9.06
−9.52
Pale yellow solid, yield 68.5%, mp 104.2–105.5°C. Rf: 0.55 (M3). FT-IR
(vmax, cm−1): 3500 (O─H); 3271 (indole NH), 3055, 2972, 2922, 2858
−9.90

(C─H); 1674, 1622, 1514, 1498, 1464 (N─H, C N, CC); 1373, 1329
−9.39
(C─H); 1209 (C─S); 1072 (pyran C─O); 792, 742 (substituted
benzene); 688 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.57 (t, 3H,
─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.27 (t, 3H, ─CH2─CH3 at C8,
J = 7.5 Hz); 1.53–2.07 (m, 2H, ─CH2─CH3 at C1); 2.26 (s, 3H, Ar─CH3);
2.53–2.90 (m, 4H, ─CH2 ─CH3 at C8 and ─CH2─ at C1); 3.31–3.47 (m,
5H, ─CH2─ at C4 and ─N─CH3); 3.77–3.95 (m, 2H, ─CH2 at C3); 4.22
(s, 2H, S─CH2); 6.87–7.25 (m, 6H, Ar─H); 10.72 (s, 1H, indole N─H).
13C NMR (150 MHz, CDCl3): δ 7.55 (C-12); 13.75 (C-10); 22.48
(N─CH3); 24.25 (C-9); 30.08 (C-4); 30.41 (C-11); 34.77 (C-13); 38.87
(S─CH2); 60.97 (C-3); 76.27 (C-1); 108.01 (C-4a); 115.73 (C-6); 119.42
(C-5); 120.22 (C-7); 126.00 (C-5a); 127.10 (C-2′ and C-6′); 128.82 (C-
4′); 129.37 (C-3′ and C-5′); 133.63 (C-8); 134.66 (C-1′); 135.83 (C-1a);
5g
5h
5i
−8.45
−9.06
−9.09
5j
−9.31
5k
5l
−11.21
−9.83
62.05
74.16
23.18
26.03
12.41
8.36
5m
5n
5p
5r
−9.73
−10.42
−10.35
−10.79
−11.02
−10.82
−11.02
5s
5t

137.64 (C-8a); 150.03 (triazole C N); 153.35 (C─S). Analysis for
11.64
7.27
C27H32N4OS.2H2O calcd. (%): C, 65.29; H, 7.31; N, 11.28; S, 6.49.
5v
Found (%): C, 65.09; H, 6.90; N, 11.41; S, 6.52.

ÇORUH ET AL.
11 of 16
|
FIGURE 6 Molecular interactions of compound 5k
(R,S)-1-{[5-(4-Nitrobenzyl)sulfanyl-4-methyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5g)
61.28; H, 6.13; N, 13.74; S, 6.29. Found (%): C, 61.24; H, 5.39; N,
13.54; S, 5.40.
Yellow solid, yield 46.4%, mp 133.4–136.8°C. Rf: 0.35 (M1). FT-IR
(vmax, cm−1): 3600 (O─H); 3190 (indole NH), 3050, 2965, 2932, 2874
(R,S)-1-{[5-Benzylsulfanyl-4-ethyl-4H-1,2,4-triazol-3-yl]methyl}-
1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (5h)
(C─H); 1600, 1518, 1479 (N─H, C N, C C); 1343, (C─H); 1250
(C─S); 1076 (pyran C─O); 745, 719 (p-substituted benzene); 698
(C─S). 1H NMR (300 MHz, CDCl3): δ 0.77 (t, 3H, ─CH2─CH3 at C1,
J = 7.2 Hz, J = 7.5 Hz); 1.31–1.38 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz,
J = 7.8 Hz); 1.99–2.29 (m, 2H, ─CH2─CH3 at C1); 2.78–2.90 (m, 4H,
─CH2 ─CH3 at C8 and ─CH2─ at C1); 3.12–3.31 (m, 5H, ─CH2─ at C4
and ─N─CH3); 3.97–4.12 (m, 2H, ─CH2 at C3); 4.40–4.50 (dd, 2H,
J = 15 Hz, J = 12 Hz, S─CH2); 6.99–7.36 (m, 3H, Ar─H); 8.11 (d, 2H,
Ar─NO2, ortho, J = 8.7 Hz); 7.49 (d, 2H, Ar─NO2, para, J = 8.7 Hz); 9.75
(s, 1H, indole N─H). 13C NMR (150 MHz, CDCl3): δ 7.60 (C-12); 13.81
(C-10); 22.39 (N─CH3); 24.26 (C-9); 30.24 (C-4); 30.75 (C-11); 34.68
(C-13); 36.89 (S─CH2); 60.94 (C-3); 76.33 (C-1); 108.41 (C-4a); 115.87
(C-6); 119.59 (C-5); 120.45 (C-7); 126.03 (C-5a); 127.37, 127.48 (C-2′
and C-6′); 128.99 (C-4′); 129.90, 130.05 (C-3′ and C-5′); 134.63 (C-8);
White solid, yield 54.8%, mp 107.7°C. Rf: 0.26 (M1). FT-IR (vmax, cm−1):


3485 (O─H); 3221 (indole N─H); 2970, 2870 (C─H); 1600, 1504,


1475, 1456, 1438 (N─H, C C, triazole C N); 1383 (C─H); 1238
(C─S); 1078 (pyran C─O); 748, 696 (mono-substituted benzene); 680
(C─S). 1H NMR (300 MHz, CDCl3): δ 0.77 (t, 3H, ─CH2─CH3 at C1,
J = 7.2 Hz, J = 7.5 Hz); 1.06 (t, 3H, triazole ─CH2─CH3, J = 7.5 Hz,
J = 7.2 Hz); 1.38 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz, J = 7.8 Hz); 2.03–
2.31 (m, 2H, ─CH2─CH3 at C1); 2.81–2.96 (m, 4H, ─CH2─CH3 at C8
and ─CH2─ at C1); 3.10–3.25 (q, 2H, triazole ─CH2─CH3); 3.62–3.7
(m, 2H, ─CH2─ at C4); 3.99–4.13 (m, 2H, ─CH2 at C3); 4.39 (s, 2H,
S─CH2); 6.99–7.37 (m, 8H, Ar─H and CHCl3); 10.41 (s, 1H, indole
N─H). 13C NMR (75 MHz, CDCl3): δ 7.64 (C-12); 13.81 (C-10); 14.89
(N─CH2─CH3); 18.48 (C-9); 22.52 (C-4); 24.31 (C-11); 30.30
(N─CH2─CH3); 34.62 (C-13); 38.79 (S─CH2); 60.99 (C-3); 76.34 (C-
1); 107.86 (C-4a); 115.76 (C-6); 119.40 (C-5); 120.20 (C-7); 126.03 (C-
5a); 127.14 (C-2′ and C-6′); 127.85 (C-4′); 128.73 (C-3′ and C-5′);

135.44 (C-1′); 144.47 (C-1a); 147.39 (C-8a); 149.33 (triazole C N);
153.69 (C─S). Analysis for C26H29N5O3S.H2O calcd. (%): C,

12 of 16
ÇORUH ET AL.
|
FIGURE 7 Molecular interactions of compound 5v


128.95 (C-8); 134.65 (C-1′); 136.01 (C-1a); 136.73 (C-8a); 149.58
152.49 (C-8a); 161.49 (triazole C N); 163.13 ( C─S). Analysis for
C27H31FN4OS.H2O calcd. (%): C, 65.33; H, 6.70; N, 11.28; S, 6.46.
Found (%): C, 65.10; H, 6.58; N, 10.94; S, 6.30.


(triazole C N); 152.65 ( C─S). C27H32N4OS.H2O calcd. (%): C,
67.75; H, 7.16; N, 11.71; S, 6.70. Found (%): C, 67.73; H, 6.89; N, 11.91;
S, 6.44.
(R,S)-1-{[5-(2-Chlorobenzyl)sulfanyl-4-ethyl-4H-1,2,4-triazole-3-
yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5j)
(R,S)-1-{[5-(4-Fluorobenzyl)sulfanyl-4-ethyl-4H-1,2,4-triazole-3-
yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5i)
White solid, yield 56.8%, mp 107.7–109.4°C. Rf: 0.47 (M3). FT-IR
(vmax, cm−1): 3532 (O─H); 3198 (indole N─H); 3087, 2961, 2920
White solid, yield 63.8%, mp 90.0–92.0°C. Rf: 0.47 (M). FT-IR
(vmax, cm−1): 3630 (O─H); 3246 (indole NH); 3022, 2970, 2931,


(C─H); 1598, 1510, 1474, 1438 (N─H, C C, triazole C N); 1377,
1350 (C─H); 1238 (C─S); 1078 (pyran C─O); 743, 694 (o-substituted
benzene); 665 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.77 (t, 3H,
─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.06 (t, 3H, triazole
─CH2─CH3, J = 7.2 Hz); 1.38 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz,
J = 7.8 Hz); 2.08–2.30 (m, 2H, ─CH2─CH3 at C1); 2.81–2.96 (m, 4H,
─CH2 ─CH3 at C8 and ─CH2─ at C1); 3.08–3.29 (q, 2H, triazole
─CH2─CH3); 3.63–3.71 (m, 2H, ─CH2─ at C4); 3.99–4.13 (m, 2H,
─CH2 at C3); 4.50–4.51 (dd, 2H, J = 15 Hz, J = 15 Hz, S─CH2); 7.07–
7.38 (m, 7H, Ar─H); 10.44 (s, 1H, indole N─H). 13C NMR (75 MHz,
CDCl3): δ 7.67 (C-12); 13.84 (C-10); 14.92 (N─CH2─CH3); 22.55 (C-9);
24.36 (C-4); 30.29 (C-11); 34.66 (N─CH2─CH3); 36.55 (C-13); 38.83
(S─CH2); 61.01 (C-3); 76.29 (C-1); 107.86 (C-4a); 115.79 (C-6); 119.41
(C-5); 120.23 (C-7); 126.06 (C-5a); 127.00 (C-2′ and C-6′); 127.11 (C-
4′); 129.38 (C-3′ and C-5′); 129.79 (C-8); 131.10 (C-1′); 134.16 (C-1a);


2850 (C─H); 1600, 1506, 1473, 1456 (N─H, C C, triazole C N);
1381, 1311 (C─H); 1211 (C─S); 1080 (pyran C─O); 790, 746 (mono-
substituted benzene); 680 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.77 (t,
3H, ─CH2─CH3 at C1, J = 7.5 Hz, J = 7.2 Hz); 1.10 (t, 3H, triazole
─CH2─CH3, J = 7.2 Hz, J = 7.5 Hz); 1.37 (t, 3H, ─CH2─CH3 at C8,
J = 7.5 Hz); 2.05–2.29 (m, 2H, ─CH2─CH3 at C1); 2.81–2.95 (m, 4H,
─CH2─CH3 at C8 and ─CH2─ at C1); 3.16–3.27 (q, 2H, triazole
─CH2─CH3); 3.67–3.74 (m, 2H, ─CH2─ at C4); 3.99–4.13 (m, 2H,
─CH2 at C3); 4.37 (s, 2H, S─CH2); 6.94–7.37 (m, 8H, Ar─H and CHCl3);
10.31 (s, 1H, indole N─H). 13C NMR (150 MHz, CDCl3): δ 7.61 (C-12);
13.79 (C-10); 14.92 (N─CH2─CH3); 22.51 (C-9); 24.31 (C-4); 30.33 (C-
11); 34.60 (N─CH2─CH3); 37.67 (C-13); 38.85 (S─CH2); 60.99 (C-3);
76.29 (C-1); 107.91 (C-4a); 115.88 (C-6); 119.42 (C-5); 120.23 (C-7);
126.03 (C-5a); 127.08, 130.65 (C-2′ and C-6′); 130.76 (C-4′); 132.59,
132.61 (C-3′ and C-5′); 134.61 (C-8); 135.97 (C-1′); 149.41 (C-1a);


134.67 (C-8a); 149.33 (triazole C N); 152.77 ( C─S). Analysis for

ÇORUH ET AL.
13 of 16
|
C27H31ClN4OS.1/2H2O calcd. (%): C, 64.33; H, 6.40; N, 11.11; S, 6.36.
Found (%): C, 64.71; H, 6.25; N, 11.00; S, 6.11.
(R,S)-1-{[5-(4-Methylbenzyl)sulfanyl-4-ethyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5m)
(R,S)-1-{[5-(4-Dichlorobenzyl)sulfanyl-4-ethyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-tetrahydropyrano[3,4-b]indole (5k)
White solid, yield 51.5%, mp 184.9–185.8°C. Rf: 0.41 (M3). FT-IR
(vmax, cm−1): 3509 (O─H); 3185 (indole N─H); 3080, 2962, 2920,
Pale yellow solid, yield 76.5%, mp 80.0–81.0°C. Rf: 0.96 (M2). FT-IR
(vmax, cm−1): 3500 (O─H); 3252 (indole N─H); 3051, 2962, 2931, 2872

(C─H); 1564, 1512, 1458, 1421 (N─H, CC, triazole C N); 1379
(C─H); 1238 (C─S); 1076 (pyran C─O); 785, 742 (mono-substituted
benzene); 694 (C─S). 1H NMR (300 MHz, CDCl3-d): δ 0.77 (t, 3H,
─CH2─CH3 at C1, J = 7.5 Hz, J = 7.2 Hz); 1.09 (t, 3H, triazole
─CH2─CH3, J = 7.5 Hz, J = 7.2 Hz); 1.38 (t, 3H, ─CH2─CH3 at C8,
J = 7.5 Hz, J = 7.8 Hz); 2.09–2.24 (m, 2H, ─CH2─CH3 at C1); 2.30 (s,
3H, Ar─CH3); 2.82–2.96 (m, 4H, ─CH2 ─CH3 at C8 and ─CH2─ at C1);
3.11–3.27 (q, 2H, triazole ─CH2─CH3); 3.64–3.72 (m, 2H, ─CH2─ at
C4); 3.99–4.14 (m, 2H, ─CH2 at C3); 4.36 (s, 2H, S─CH2); 6.99–7.37 (m,
8H, Ar─H ve CHCl3); 10.42 (s, 1H, indole N─H). Analysis for

2872, 2860 (C─H); 1609, 1491, 1471, 1435 (N─H, C C, triazole

C
N); 1411, 1377 (C─H); 1236 (C─S); 1078 (pyran C─O); 742, 723
(p-substituted benzene); 641 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.77
(t, 3H, ─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.10 (t, 3H, triazole
─CH2─CH3, J = 7.2 Hz, J = 7.5 Hz); 1.37 (t, 3H,─CH2─CH3 at C8,
J = 7.5 Hz); 2.07–2.30 (m, 2H, ─CH2─CH3 at C1); 2.83–2.95 (m, 4H,
─CH2─CH3 at C8 and ─CH2─ at C1); 3.11–3.26 (q, 2H, triazole
─CH2─CH3); 3.66–3.74 (m, 2H, ─CH2─ at C4); 3.99–4.12 (m, 2H,
─CH2 at C3); 4.32–4.42 (dd, 2H, J = 12 Hz, J = 12 Hz, S─CH2); 6.99–
7.37 (m, 8H, Ar─H and CHCl3); 10.32 (s, 1H, indole N─H). 13C NMR
(75 MHz, CDCl3): δ 7.60 (C-12); 13.79 (C-10); 14.93 (N─CH2─CH3);
22.51 (C-9); 24.31 (C-4); 30.33 (C-11); 34.60 (N─CH2─CH3); 37.61
(C-13); 38.86 (S─CH2); 60.99 (C-3); 76.28 (C-1); 107.92 (C-4a); 115.78
(C-6); 119.42 (C-5); 120.24 (C-7); 126.03 (C-5a); 127.07 (C-2′ and C-
6′); 128.85 (C-4′); 130.33 (C-3′ and C-5′); 133.73 (C-8); 134.60 (C-1′);
C28H34N4OS.2H2O calcd. (%): C, 65.85; H, 7.50; N, 10.97; S, 6.28.
Found (%): C, 66.34; H, 7.07; N, 11.53; S, 6.39.
(R,S)-1-{[5-Benzylsulfanyl-4-phenyl-4H-1,2,4-triazole-3-yl]-
methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (5n)
White solid, yield 87.5%, mp 120.0–122.0°C. Rf: 0.14 (M3). FT-IR
(vmax, cm−1): 3219 (indole N─H); 3059, 2962, 2929, 2872 (C─H); 1597,



135.34 (C-1a); 135.94 (C-8a); 149.27 (triazole CN); 152.72 ( C─S).
1496, 1452, 1444 (N─H, C N, C C); 1369, 1336 (C─H); 1236 (C─S);
1076 (pyran C─O); 765, 747 (mono-substituted benzene); 694 (C─S).
1H NMR (300 MHz, CDCl3): δ 0.71 (t, 3H, ─CH2─CH3 at C1, J = 7.5 Hz,
J = 7.2 Hz); 1.41 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz, J = 7.2 Hz); 2.10–
2.30 (m, 2H, ─CH2─CH3 at C1); 2.65–2.98 (m, 4H, ─CH2 ─CH3 at C8
and CH2─ at C1); 3.09, 3.12 (d, 2H, ─CH2─ at C4); 3.68–3.93 (m, 4H,
─CH2 at C3); 4.38–4.43 (dd, 2H, J = 12.6 Hz, J = 12.6 Hz, S─CH2);
7.01–7.46 (m, 13H, Ar─H); 10.11 (s, 1H, indole N─H). 13C NMR
(75 MHz, CDCl3): δ 7.56 (C-12); 13.80 (C-10); 22.38 (C-9); 24.29
(C-4); 30.46 (C-11); 34.50 (C-13); 37.63 (S─CH2); 60.74 (C-3); 76.36
(C-1); 108.08 (C-4a); 115.77 (C-6); 119.47 (C-5); 120.25 (C-7); 126.06
(C-5a); 127.06, 127.2, 127.80, 128.68 (C-2′ and C-6′); 129.13,
129.88 (C-4′); 130.14 (C-3′ and C-5′); 132.75 (C-8); 134.62 (C-1′);
HR MS [(EI+), m/z (%)]: 496.1864 ([M+2]+, 2.98), 494.1882 ([M+], 7.24),
228.1369 (100.0), 227.1293 (16.29), 142.0461 (2.92), 125.0150
(7.05), 57.0333 (4.04). Analysis for C27H31ClN4OS.1/2H2O calcd.
(%): C, 64.33; H, 6.40; N, 11.11; S, 6.36. Found (%): C, 64.10; H, 6.14; N,
10.89; S, 5.60.
(R,S)-1-{[5-(2,6-Dichlorobenzyl)sulfanyl-4-ethyl-4H-1,2,4-
triazole-3-yl]methyl}-1,8-diethyl-tetrahydropyrano[3,4-b]indole
(5l)
White solid, yield 42.2%, mp 106.0–107.0°C. Rf: 0.25 (M3). FT-IR
(vmax, cm−1): 3510 (O─H); 3213 (indole N─H); 3011, 2965, 2922, 2872


(C─H); 1579, 1562, 1510, 1473, 1437 (N─H, C C, triazole C N);
1375, 1348 (C─H); 1236 (C─S); 1076 (pyran C─O); 779, 762, 742
(substituted benzene); 677 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.79 (t,
3H, ─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.16 (t, 3H, triazole
─CH2─CH3, J = 7.5 Hz, J = 7.2 Hz); 1.37 (t, 3H, ─CH2─CH3 at C8,
J = 7.5 Hz, J = 7.8 Hz); 2.10–2.30 (m, 2H, ─CH2─CH3 at C1); 2.87–2.95
(m, 4H, ─CH2 ─CH3 at C8 and ─CH2─ at C1); 3.14–3.29 (q, 2H, triazole
─CH2─CH3); 3.74–3.82 (m, 2H, ─CH2─ at C4); 4.03–4.13 (m, 2H,
─CH2 at C3); 4.60–4.70 (dd, 2H, J = 12 Hz, J = 12 Hz, S─CH2); 7.01–
7.37 (m, 7H, Ar─H); 10.55 (s, 1H, indole N─H). 13C NMR (75 MHz,
CDCl3): δ 7.63 (C-12); 13.73 (C-10); 15.14 (N─CH2─CH3); 22.52 (C-9);
24.26 (C-4); 30.06 (C-11); 34.75 (N─CH2─CH3); 34.97 (C-13); 38.99
(S─CH2); 60.99 (C-3); 76.29 (C-1); 107.66 (C-4a); 115.69 (C-6); 119.34
(C-5); 120.11 (C-7); 126.00 (C-5a); 127.18, 128.47 (C-2′ and C-6′);
128.54 (C-4′); 129.52, 130.25 (C-3′ and C-5′); 132.88 (C-8); 134.62 (C-


135.87 (C-1a); 136.32 (C-8a); 151.31 (triazol C N); 153.46 ( C─S).
HR MS [(EI+), m/z (%)]: 508.2284 ([M+], 7.81), 281.0976 (5.50),
229.1413 (16.90), 228.1377 (100.0), 227.1300 (9.88), 91.0543 (8.00),
57.0335 (3.96). Analysis for C31H32N4OS.1/2H2O calcd. (%): C, 71.92;
H, 6.43; N, 10.82; S, 6.19. Found (%): C, 71.78; H, 6.43; N, 10.60;
S, 5.94.
(R,S)-1-{[5-(4-Fluorobenzyl)sulfanyl-4-phenyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5p)
White solid, yield 56.8%, mp 98.1°C. Rf: 0.34 (M1). FT-IR (vmax, cm−1):
3446 (O─H); 3223 (indole N─H); 3016, 2962, 2950, 2853 (C─H);


1597, 1510, 1497, 1447, 1424 (N─H, C C, triazole C N); 1338
(C─H); 1223 (C─S); 1076 (pyran C─O); 748, 692 (p-substituted
benzene); 664 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.71 (t, 3H,
─CH2─CH3 at C1, J = 7.2 Hz); 1.40 (t, 3H, ─CH2─CH3 at C8, J = 7.5 Hz,
J = 7.8 Hz); 2.01–2.31 (m, 2H, ─CH2─CH3 at C1); 2.59–3.15
(m, 6H, ─CH2 ─CH3 at C8 and ─CH2 at C1 and ─CH2─ at C4);

1′); 135.86 (C-1a); 1366.14 (C-8a); 48.85 (triazole C N); 152.91

(
C─S). Analysis for C27H30Cl2N4OS.1/2H2O calcd. (%): C,
60.22; H, 5.80; N, 10.40; S, 5.95. Found (%): C, 59.44; H, 5.86; N,
9.91; S, 5.43.

14 of 16
ÇORUH ET AL.
|
3.61–3.99 (m, 2H, ─CH2 at C3); 4.38 (s, 2H, S─CH2); 6.92–7.50 (m,
12H, Ar─H); 9.95 (s, 1H, indole N─H). 13C NMR (75 MHz, CDCl3): δ
7.52 (C-12); 13.77 (C-10); 22.35 (C-9); 24.26 (C-4); 30.54 (C-11); 34.47
(C-13); 36.54 (S-CH2); 60.71 (C-3); 76.33 (C-1); 108.17 (C-4a); 115.45
(C-6); 119.48 (C-5); 120.28 (C-7); 126.05 (C-5a); 126.98, 127.20,
129.91, 130.19 (C-2′ and C-6′); 130.78, 130.83 (C-4′); 132.18, 132.20
(C-3′ and C-5′); 134.59 (C-8); 135.75 (C-1′); 151.12 (C-1a);
(R,S)-1-{[5-(4-Methylbenzyl)sulfanyl-4-phenyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5t)
White solid, yield 56.3%, mp 126.8°C. Rf: 0.49 (M1). FT-IR (vmax, cm−1):
3426 (O─H); 3225 (indole N─H); 3035, 2965, 2934, 2874 (C─H);


1595, 1516, 1497, 1445, 1425 (N─H, C C, triazole C N); 1337
(C─H); 1240 (C─S); 1076 (pyran C─O); 746, 692 (p-substituted
benzene); 665 (C─S). 1H NMR (300 MHz, CDCl3): δ 0.71 (t, 3H,
─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz); 1.24 (t, 3H, OH─CH2─CH3,
J = 6.9 Hz, J = 7.2 Hz); 1.41 (t, 3H, ─CH2─CH3 at C8 J = 7.5 Hz); 2.04–
2.28 (m, 2H, ─CH2─CH3 at C1); 2.30 (s, 3H, Ar─CH3); 2.62–3.14 (m,
6H, ─CH2 ─CH3, at C8 and ─CH2 at C1 and ─CH2─ at C4); 3.63–3.93
(m, 2H, ─CH2 at C3); 4.33–4.42 (dd, 2H, J = 12 Hz, J = 12 Hz, S─CH2);
7.00–7.48 (m, 12H, Ar─H); 9.96 (s, 1H, indole N─H). 13C NMR
(75 MHz, CDCl3): δ 7.52 (C-12); 13.76 (C-10); 22.37 (C-9); 24.27 (C-4);
30.43 (C-11); 34.51 (C-13); 37.29 (S-CH2); 60.73 (C-3); 76.35 (C-1);
108.03 (C-4a); 115.74 (C-6); 119.44 (C-5); 120.21 (C-7); 126.04 (C-5a);
127.05, 127.26, 129.02, 129.31 (C-2′ and C-6′); 129.84 (C-4′); 130.09,
132.78 (C-3′ and C-5′); 133.14 (C-8); 134.61 (C-1′); 135.89 (C-1a);


153.50 (C-8a); 161.46 (triazol C N); 163.10 ( C─S). Analysis for
C31H31FN4OS.2H2O calcd. (%): C, 66.17; H, 6.27; N, 9.96; S, 5.70.
Found (%): C, 66.11; H, 6.23; N, 10.03; S, 5.47.
(R,S)-1-{[5-(4-Chlorobenzyl)sulfanyl-4-phenyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahidropirano[3,4-b]indole
(5r)
White solid, yield 68.2%, mp 100.0–102.0°C. Rf: 0.37 (M1). FT-IR
(vmax, cm−1): 3510 (O─H); 3184 (indole N─H); 3015, 2963, 2930, 2872


(C─H); 1597, 1491, 1444, 1423 (N─H, C C, triazole C N); 1371, 1339
(C─H); 1238 (C─S); 1076 (pyran C─O); 745, 694 (p-substituted benzene).
1H NMR (300 MHz, CDCl3): δ 0.71 (t, 3H, ─CH2─CH3 at C1, J = 7.2 Hz,
J = 7.5 Hz); 1.40 (t, 3H, ─CH2─CH3 at C8, J = 7.2 Hz, J = 7.8 Hz); 2.02–
2.31 (m, 2H, ─CH2─CH3 at C1); 2.76–3.14 (m, 6H, ─CH2 ─CH3 at C8 and
─CH2 at C1 and ─CH2─at C4); 3.59–3.95 (m, 2H, ─CH2 at C3); 4.36 (s, 2H,
S─CH2); 7.00–7.48 (m, 13H, Ar─H and CHCl3); 9.96 (s, 1H, indole N─H).
13CNMR(75 MHz, CDCl3): δ7.54 (C-12); 13.80 (C-10); 22.36 (C-9); 24.29
(C-4); 30.58 (C-11); 34.49 (C-13); 36.57 (S─CH2); 60.73 (C-3); 76.34 (C-1);
108.20 (C-4a); 115.80 (C-6); 119.50 (C-5); 120.31 (C-7); 126.07 (C-5a);
126.99, 127.20, 128.79, 129.93 (C-2′ and C-6′); 130.22, 130.48 (C-4′);
132.66, 133.66 (C-3′ and C-5′); 134.60 (C-8); 135.04 (C-1′); 135.74 (C-


137.57 (C-8a); 151.44 (triazole C N); 153.37 ( C─S). Analysis for
C32H34N4OS.CH3CH2OH.H2O calcd. (%): C, 69.59; H, 7.210; N, 9.55;
S, 5.46. Found (%): C, 69.19; H, 6.75; N, 10.27; S, 5.57.
(R,S)-1-{[5-(4-Nitrobenzyl)sulfanyl-4-phenyl-4H-1,2,4-triazole-
3-yl]methyl}-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole
(5v)
Yellow solid, yield 48.5%, mp 88.0–91.0°C. Rf: 0.23 (M1). FT-IR
(vmax, cm−1): 3225 (indole N─H); 3025, 2963, 2930, 2872 (C─H);




1a); 150.98 (C-8a); 153.57 (triazol C N); 163.10 ( C─S). Analysis for
1599, 1518, 1497, 1445 (N─H, C C, triazole C N); 1343, 1304
(C─H); 1238 (C─S); 1076 (pyran C─O); 745, 694 (p-substituted
benzene). 1H NMR (300 MHz, CDCl3): δ 0.72 (t, 3H, ─CH2─CH3 at
C1 J = 7.2 Hz); 1.39 (t, 3H, ─CH2─CH3 at C8 J = 7.5 Hz); 2.05–2.33 (m,
2H, ─CH2─CH3 at C1); 2.75–3.27 (m, 6H, ─CH2 ─CH3, at C8 and ─CH2
at C1 and ─CH2─ at C4); 3.61–3.93 (m, 2H, ─CH2 at C3); 4.43–4.53 (dd,
2H, J = 15 Hz, J = 15 Hz, S─CH2); 6.83–7.48 (m, 8H, Ar─H); 8.13 (d, 2H,
Ar─NO2, ortho, J = 8.7 Hz); 7.53 (d, 2H, Ar─NO2, para, J = 8.7 Hz); 9.775
(s, 1H, indole N─H). 13C NMR (75 MHz, CDCl3): δ 7.53 (C-12); 13.80 (C-
10);22.31(C-9);24.28(C-4); 30.72 (C-11);34.42 (C-13);35.85(S─CH2);
60.69 (C-3); 76.30 (C-1); 108.36 (C-4a); 115.84 (C-6); 119.55 (C-5);
120.40 (C-7); 123.79 (C-5a); 126.05, 126.86, 127.10, 130.03 (C-2′ and
C-6′);130.37 (C-4′);132.46 (C-3′ and C-5′);134.54 (C-8); 135.51(C-1′);
C
31H31ClN4OS.CH3CH2OH calcd. (%): C, 67.27; H, 6.33; N, 9.51; S, 5.44.
Found (%): C, 67.26; H, 5.74; N, 9.06; S, 4.88.
(R,S)-1-{[5-(2,6-Dichlorobenzyl)sulfanyl-4-phenyl-4H-1,2,4-
triazole-3-yl]methyl}-1,8-diethyl-tetrahydropyrano[3,4-b]indole
(5s)
White solid, yield 88.0%, mp 85.0–86.0°C. Rf:0.71(M3). FT-IR (vmax,cm−1):
3400 (O─H), 3210 (indole N─H); 3072, 2963, 2927, 2872 (C─H); 1560,


1498, 1435 (N─H, C N, C C); 1368, 1336 (C─H); 1239 (C─S); 1076
(pyran C─O); 765, 747 (mono-substituted benzene); 693 (C─S). 1H NMR
(300 MHz, CDCl3): δ 0.72 (t, 3H, ─CH2─CH3 at C1, J = 7.2 Hz, J = 7.5 Hz);
1.22–1.27 (t, 3H, OH─CH2─CH3, J = 6.9 Hz); 1.38–1.43 (t, 3H,
─CH2─CH3 at C8, J = 7.5 Hz); 2.07–2.28 (m, 2H, ─CH2─CH3 at C1);
2.65–3.19 (m, 6H, ─CH2 ─CH3 at C8, ─CH2─ at C1 and OH─CH2─CH3);
3.67–3.94 (m, 4H, ─CH2─ at C4 and ─CH2 at C3); 4.62–4.70 (dd, 2H,
J = 12 Hz, J = 12 Hz, S─CH2); 7.08–7.49 (m, 12H, Ar─H); 10.18 (s, 1H,
indole N─H). 13C NMR (75 MHz, CDCl3): δ 7.54 (C-12); 13.75 (C-10);
22.38(C-9); 24.25 (C-4);30.35 (C-11); 33.78(C-13); 34.65 (S─CH2); 60.75
(C-3); 76.37 (C-1); 107.98 (C-4a); 115.72 (C-6); 119.43 (C-5); 120.18 (C-7);
126.03 (C-5a); 127.10, 127.24, 128.40, 129.47 (C-2′and C-6′); 129.89 (C-
4′); 130.11, 132.25 (C-3′ and C-5′); 132.82 (C-8); 134.63 (C-1′); 135.93


144.29 (C-1a); 147.36 (C-8a); 150.45 (triazole C N); 153.80 ( C─S).
Analysis for C31H31N5O3S.H2O calcd. (%): C, 65.13; H, 5.82; N, 12.25; S,
5.61. Found (%): C, 65.91; H, 5.76; N, 12.23; S, 5.38.
|
4.2 Biological assays
|
4.2.1 Cell culture and cell viability assay
Cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Monkey kidney epithelial
cell line VERO, human ovarian cancer cell line SKOV3, human breast
cancer cell line MCF7, human hepatocellular cancer cell line HEPG2,

(C-1a); 136.04 (C-8a); 150.56 (triazole CN); 153.77 ( C─S). Analysis for
C
31H30Cl2N4OS. CH3CH2OH.H2O calcd. (%): C, 61.77; H, 5.97; N, 8.73; S,
5.00. Found (%): C, 61.52; H, 5.73; N, 8.60; S, 5.40.

ÇORUH ET AL.
15 of 16
|
human prostate cancer cell line PC3 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supple-
mented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and
100 μg/mL streptomycin and kept in a humidified atmosphere at
37°C incubator with 5% CO2 in air. To determine the cell viability
cells were plated onto 96-well plates (1 × 104 cells/well). The cells
were treated with different concentrations (0, 10, 50, 100, 200, and
500 μM) of synthesized compound (5a–v) derivatives from etodolac
and incubated for 24 h. After the incubation cells were washed with
PBS and added to 100 μL DMEM. A total of 10 μL of the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Vy-
brant, Invitrogen) labeling reagent was added to each well and
incubated for 4 h in humidified atmosphere at 37°C incubator with
5% CO2 in air. After the incubation, 100 μL of the SDS buffer was
added into each well for solubilization of formazan precipitate. Then
absorbance was measured by microplate reader at 570 nm and was
carried out in triplicate of each assay.[18]
Similarly, PC3 cells were incubated with 10 μM concentrations of
5k, 5s, and 5v at different times (0, 12, 24, 48 h) in 12-well plates. After
the incubation, cells were harvested with cell lysis buffer. Caspase-3
activity assay was performed with caspase-3 activity assay kit
according to the manufacturer's instructions (Invitrogen).
|
4.2.5 Western blot analysis
SKOV3 cells were homogenized in cell lysis buffer and protein
concentrations were determined by Bradford reagent (Biorad).
Proteins were seperated by SDS-PAGE and transferred onto
PVDF membrane (Santa Cruz). Membrane was blocked with 3%
BSA at room temperature and was incubated with primary
antibodies. The following primary antibodies at 4°C overnight:
anti-Caspase-3 (1:500), anti-Caspase-8 (1:500), anti-Caspase-9
(1:500) from Santa Cruz. Then incubated with a primary antibody,
membrane was incubated with a horseradish peroxidase-conjugated
secondary antibodies for room temperature. Bands were visualized
by the chemiluminescence Western blotting detection reagent in
imaging system. β-Actin protein levels were used as a control to
verify equal protein loading.
|
4.2.2 Cell survival assays
In order to investigate the effects of compounds on cell survival of
SKOV3, HEPG2, MCF7, VERO cells, we treated cells with selected
etodolac derivatives of 5d and 5h at a final concentration of 10 μM in
growth medium (without antibiotics) for 24 h. After the incubation,
methylene blue staining was used for cell survivals. The cells were
extracted with 1% SDS in PBS solution and stained with methylene
blue solutions. The absorbance was measured at 600 nm with
microplate reader.[19]
|
4.3 Molecular docking
The preparation of the protein human MetAP2 as well as compounds
was performed in BIOVIA Discovery Studio 4.5. The X-ray crystal
structure of the receptor was downloaded from PDB database-
accession code: 1QZY, resolution: 1.60 A (http://www.rcsb.org/pdb/).
The protein structure was cleaned from inhibitor, water molecules, and
non-interacting ions. All hydrogens were added and protein was
minimized using “Clean Geometry” toolkit, then for more complete
optimization it was submitted to “Prepare Macromolecule” protocol.
Missing hydrogen atoms were added based on the protonation state of
the titratable residues at a pH of 7.4. The CHARMm force field was
assigned.
|
4.2.3 Determination of apoptosis
SKOV3 ovarian cancer cells were treated with 10 μM concentration of
etodolac and derivatives 5d and 5h at different times (0, 6, 12, 24 h) in
12-well plates at density of 2 × 104 cells/well in triplicate. Cell growing
was imaged using a Zeiss Axio inverted microscope (10×) imaging
system. Methylene blue staining was used for quantification for cell
growing.
Compounds were drawn using “small molecules” toolkit and
minimized using the same force field. After preparation docking was
performed in AutoDock 4.2 programme (http://autodock.scripps.edu).
All the compounds were set to be flexible, and protein is set to be rigid.
AutoDock adds a free-energy scoring function created from a linear
regression analysis, the AMBER force field, and a large set of diverse
protein-ligand complexes with known inhibition constants. For the
center of binding pocked (grid box) HIS231 was used. The size grid box
was set to be 60, 60, and 60. The searching algorithms include
Lamarckian genetic algorithm.
Apoptosis in the SKOV3 ovarian cancer cell line was measured
using Acridine orange and ethidium bromide (AO/EB). After 24 h
incubation with 10 μM concentrations of etodolac and derivatives in
12-well plates at density of 2 × 105 cells/well in triplicate. Cells were
then stained for 1 μg/mL AO/EB solutions and the fluorescent
intensities specific of followed by microscope (Zeiss). Living cells
grouped with green intensity and apoptotic cells grouped with red
intensity.
For visualization of non-bonded interactions BIOVIA, and creating
the pictures Discovery Studio 4.5 was used.
|
4.2.4 Measurement of caspase-3 activity
SKOV3 cells were incubated with 10 μM concentrations of 5d and 5h
at different times (0, 6, 12, 24 h) in 12-well plates. After the
incubations, cells were harvested with cell lysis buffer. Caspase-3
activity assay was performed with caspase-3 activity assay kit
according to the manufacturer's instructions (Invitrogen).[20]
ACKNOWLEDGMENTS
This study has been financially supported by the Scientific Research
Project Commission of Marmara University (Project number: SAG-C-
DRP-041213-0451). The authors are grateful to Jürgen Gross from the

16 of 16
ÇORUH ET AL.
|
Institute of Organic Chemistry, University of Heidelberg, for his
generous help in obtaining HR-EI mass spectra of the synthesized
compounds.
[11] B. Kummari, N. Polkam, P. Ramesh, H. Anantaraju, P. Yogeeswari,
J. S. Anireddy, S. D. Guggilapud, B. N. Babud, RSC Adv. 2017,
7, 23680.
[12] İ. Küçükgüzel, Ş. G. Küçükgüzel, S. Rollas, G. Ötük-Sanış, O. Özdemir,
İ. Bayrak, T. Altuğ, J. P. Stables, Il Farmaco. 2004, 59(11), 893.
[13] İ. Küçükgüzel, Ş. G. Küçükgüzel, S. Rollas, M. Kiraz, Bioorg. Med. Chem.
Lett. 2001, 11(13), 1703.
CONFLICT OF INTEREST
[14] K. Zamani, K. Faghihi, T. Tofighi, M. R. Shariatzadeh, Turk. J. Chem.
2004, 28, 95.
The authors have declared no conflicts of interest.
[15] L. Popiolek, U. Kosikowska, L. Mazur, M. Dobosz, A. Malm, Med.
Chem. Res. 2013, 22, 3134.
ORCID
[16] G. Tu, Y. Yan, X. Chen, Q. Lv, J Wang, S. Li, Drug Discov. Ther. 2013,
7(2), 58.
Ş. Güniz Küçükgüzel
http://orcid.org/0000-0001-9405-8905
[17] N. Ulusoy, G. Çapan, N. Ergenç, A. C. Ekinci, A. Vidin, Acta Pharm.
Turcica 1998, 1, 5.
[18] S. H. Baek, Y. O. Kim, J. S. Kwag, K. E. Choi, W. Y. Jung, D. S. Han, Arch.
Pharm. Res. 1998, 21, 353.
REFERENCES
[19] A. H. Sheila, S. K. Frank, TCA Manual/Tissue Culture Association 1975,
1(2), 103.
[1] Ş. G. Küçükgüzel, P. Süzgün, Eur. J. Med. Chem. 2015, 97, 830.
[2] Y.-P. Hou, J. Sun, Z.-H. Pang, P.-C. Lv, D.-D. Li, L. Yan, H.-J. Zhang,
E. X. Zheng, J. Zhao, H.-L. Zhu, Bioorg. Med. Chem. 2011, 19, 5948.
[3] S.-Q. Yin, R. A. Jr. Galemmo, Z.-P. Liu, Curr. Med. Chem. 2012, 19,
1021.
[20] H. Jaeschke, M. A. Fisher, J. A. Lawson, C. A. Simmons, A. Farhood,
D. A. Jones, J. Immunol. 1998, 160(7), 3480.
[21] H. Towbin, K. W. Bair, J. A. DeCaprio, M. J. Eck, S. Kim, F. R. Kinder, A.
Morollo, D. R. Mueller, P. Schindler, H. K. Song, J. van Oostrum, R. W.
Versace, H. Voshol, J. Wood, S. Zabludoff, P. E. Phillips, J. Biol. Chem.
2003, 278(52), 52964.
[4] T. Garrabrant, R. W. Tuman, D. Ludovici, R. Tominovich, R. L.
Simoneaux, R. A. Galemmo, Jr., D. L. Johnson, Angiogenesis 2004, 7,
91.
[5] J. P. Jr. Marino, P. W. Fisher, G. A. Hofmann, R. B. Kirkpatrick, C. A.
Janson, R. K. Johnson, C. Ma, M. Mattern, T. D. Meek, M. D. Ryan, C.
Schulz, W. W. Smith, D.G Tew, T. A. Jr. Tomazek, D. F Veber, W. C.
Xiong, Y. Yamamoto, K. Yamashita, G. Yang, S. K. Thompson, J. Med.
Chem. 2007, 50, 3777.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[6] M. Kobayashi, S. Nakamura, K. Shibata, N. Sahara, K. Shigeno, K.
Shinjo, K. Naito, K. Ohnishi, Eur. J. Haematol. 2005, 75(3), 212.
[7] S. Murata, M. Adachi, M. Kioi, S. Torigoe, K. Ijichi, Y. Hasegawa, T.
Ogawa, M. K. Bhayani, S. Y. Lai, K. Mitsudo, I. Tohnai, Anticancer Res.
2011, 31(9), 2893.
How to cite this article: Çoruh I, Çevik Ö, Yelekçi K, Djikic T,
Küçükgüzel ŞG. Synthesis, anticancer activity, and molecular
modeling of etodolac-thioether derivatives as potent
methionine aminopeptidase (type II) inhibitors. Arch Pharm
Chem Life Sci. 2018;351:e1700195.
[8] P. Çıkla, D. Özsavcı, Ö. Bingöl-Özakpınar, A. Şener, Ö. Çevik, S.
Özbaş-Turan, J. Akbuğa, F. Şahin, Ş. G. Küçükgüzel, Arch. Pharm.
2013, 346, 367.
[9] P. Çıkla, P. Arora, A. Basu, T. T. Talele, N. Kaushik-Basu, Ş. G.
Küçükgüzel, Marmara Pharm. J. 2013, 17, 138.
[10] P. Çıkla-Süzgün, N. Kaushik-Basu, A. Basu, P. Arora, T.T. Talele,
İ. Durmaz, R. Cetin-Atalay, Ş. G. Kucukguzel, J. Enzyme Inhib. Med.
Chem. 2015, 30(5), 778.
https://doi.org/10.1002/ardp.201700195