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

Received: 7 September 2017

Revised: 4 February 2018

Accepted: 6 February 2018

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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

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Ş. Güniz Küçükgüzel¹

¹Faculty 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]-

²Department 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 10⁻⁵M (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.

³Faculty 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.

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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.

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Arch Pharm Chem Life Sci. 2018;351:e1700195.

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ÇORUH ET AL.

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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⁻¹which 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−

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−

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

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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−

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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−

5v

C₆H₅−

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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-CH₂protons.^[15–17]Mostly, the S-CH₂protons 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,

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2.2 Biological evaluation

chemical shifts of the S-CH₂appeared upfield at 39.16, 37.90, 34.97,



38.79, 38.83, and 37.63 ppm, respectively, C N appeared downfield

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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),

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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 IC₅₀values

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 IC₅₀of 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).

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TABLE 2 IC₅₀values 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

─NO₂group 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 K_i= 6.10 nM, com-

pound 5s with ΔG = −11.02 kcal/mol and K_i= 8.36 nM, and 5v

11.02 kcal/mol and K_i= 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

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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 IC₅₀values 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

IC₅₀levels 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

10⁻⁵M 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 IC₅₀values of 7.22

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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 IC₅₀values 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 CDCl₃and DMSO-d₆on 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 CDCl₃operating 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, K_i= 6.10 nM, 5s with the values of

ΔG = −11.02 kcal/mol, K_i= 8.36 nM, and 5v with the values of

ΔG = −11.02 kcal/mol, K_i= 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.

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4

EXPERIMENTAL

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4.1 Chemistry

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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.

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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)

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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.

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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. R_f: 0.37 (M₁). FT-IR

(v_max, cm⁻¹): 3182 (indole N─H); 3084, 3028, 2964, 2928, 2860 (C─H);

``Pale yellow solid, yield 47.30%, mp 161.1°C. R_f: 0.10 (M₁). FT-IR

(v_max, 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─CH₃at C₁, J = 7.2 Hz, J = 7.8 Hz); 1.27 (t, 3H, ─CH₂─CH₃at

C₈, J = 7.2 Hz, J = 7.8 Hz); 1.56–2.07 (m, 2H, ─CH₂─CH₃at C₁); 2.50–

2.89 (m, 4H, ─CH₂─CH₃at C₈, ─CH₂at C₁); 3.31–3.44 (m, 5H, ─CH₂

at C₄and N─CH₃); 3.78–3.90 (m, 2H, ─CH₂at 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─CH₃at

C₁, J = 7.2 Hz); 1.37 (t, 3H, ─CH₂─CH₃at C₈, J = 7.5 Hz, J = 7.8 Hz);

1.98–2.3 (m, 2H, ─CH₂─CH₃at C₁); 2.80–2.93 (m, 4H, ─CH₂─CH₃

at C₈and ─CH₂─ at C₁); 3.09–3.25 (m, 5H, ─CH₂─ at C₄and

─N─CH₃); 3.98–4.13 (m, 2H, ─CH₂at 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.

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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. R_f: 0.19 (M₁). FT-IR (v_max, 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─CH₃at C₁, J = 7.2 Hz, J = 7.5 Hz); 1.37 (t, 3H, ─CH₂─CH₃at

C₈, J = 7.5 Hz, J = 7.8 Hz); 1.97–2.29 (m, 2H, ─CH₂─CH₃at C₁); 2.79–

2.94 (m, 4H, ─CH₂─CH₃at C₈and ─CH₂─ at C₁); 3.07–3.24 (m, 5H,

─CH₂─ at C₄and ─N─CH₃); 3.98–4.13 (m, 2H, ─CH₂at 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. R_f: 0.53 (M₁). FT-IR

(v_max, 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─CH₃at C₁, J = 7.5 Hz, J = 7.2 Hz); 1.36 (t,

3H, ─CH₂─CH₃at C₈, J = 7.5 Hz, J = 7.8 Hz); 1.98–2.30 (m, 2H,

─CH₂─CH₃at C₁); 2.76–2.92 (m, 4H, ─CH₂─CH₃at C₈and ─CH₂─ at

C₁); 3.14–3.25 (m, 5H, ─CH₂─ at C₄and ─N─CH₃); 3.98–4.13 (m, 2H,

─CH₂at 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.

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TABLE 3 IC₅₀values 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. R_f: 0.57 (M₂). FT-IR

(v_max, 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─CH₃at C₁, J = 7.2 Hz,

J = 7.5 Hz); 1.35 (t, 3H, ─CH₂─CH₃at C₈, J = 7.8 Hz, J = 7.5 Hz); 1.98–

2.26 (m, 2H, ─CH₂─CH₃at C₁); 2.81–2.92 (m, 4H, ─CH₂─CH₃at C₈

and ─CH₂─ at C₁); 3.11–3.28 (m, 5H, ─CH₂─ at C₄and ─N─CH₃);

4.01–4.12 (m, 2H, ─CH₂at 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

K_i(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. R_f: 0.55 (M₃). FT-IR

(v_max, 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─CH₃at C₁, J = 7.2 Hz, J = 7.5 Hz); 1.27 (t, 3H, ─CH₂─CH₃at C₈,

J = 7.5 Hz); 1.53–2.07 (m, 2H, ─CH₂─CH₃at C₁); 2.26 (s, 3H, Ar─CH₃);

2.53–2.90 (m, 4H, ─CH₂─CH₃at C₈and ─CH₂─ at C₁); 3.31–3.47 (m,

5H, ─CH₂─ at C₄and ─N─CH₃); 3.77–3.95 (m, 2H, ─CH₂at 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.

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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. R_f: 0.35 (M₁). FT-IR

(v_max, 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─CH₃at C₁,

J = 7.2 Hz, J = 7.5 Hz); 1.31–1.38 (t, 3H, ─CH₂─CH₃at C₈, J = 7.5 Hz,

J = 7.8 Hz); 1.99–2.29 (m, 2H, ─CH₂─CH₃at C₁); 2.78–2.90 (m, 4H,

─CH₂─CH₃at C₈and ─CH₂─ at C₁); 3.12–3.31 (m, 5H, ─CH₂─ at C₄

and ─N─CH₃); 3.97–4.12 (m, 2H, ─CH₂at 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. R_f: 0.26 (M₁). FT-IR (v_max, 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─CH₃at 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─CH₃at C₈, J = 7.5 Hz, J = 7.8 Hz); 2.03–

2.31 (m, 2H, ─CH₂─CH₃at C₁); 2.81–2.96 (m, 4H, ─CH₂─CH₃at 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, ─CH₂at 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,

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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. R_f: 0.47 (M₃). FT-IR

(v_max, cm⁻¹): 3532 (O─H); 3198 (indole N─H); 3087, 2961, 2920

White solid, yield 63.8%, mp 90.0–92.0°C. R_f: 0.47 (M). FT-IR

(v_max, 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─CH₃at 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─CH₃at C₈, J = 7.5 Hz,

J = 7.8 Hz); 2.08–2.30 (m, 2H, ─CH₂─CH₃at C₁); 2.81–2.96 (m, 4H,

─CH₂─CH₃at C₈and ─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,

─CH₂at 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─CH₃at 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─CH₃at C₈,

J = 7.5 Hz); 2.05–2.29 (m, 2H, ─CH₂─CH₃at C₁); 2.81–2.95 (m, 4H,

─CH₂─CH₃at C₈and ─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,

─CH₂at 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.

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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. R_f: 0.41 (M₃). FT-IR

(v_max, cm⁻¹): 3509 (O─H); 3185 (indole N─H); 3080, 2962, 2920,

Pale yellow solid, yield 76.5%, mp 80.0–81.0°C. R_f: 0.96 (M₂). FT-IR

(v_max, 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─CH₃at 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─CH₃at C₈,

J = 7.5 Hz, J = 7.8 Hz); 2.09–2.24 (m, 2H, ─CH₂─CH₃at C₁); 2.30 (s,

3H, Ar─CH₃); 2.82–2.96 (m, 4H, ─CH₂─CH₃at C₈and ─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, ─CH₂at 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─CH₃at 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─CH₃at C₈,

J = 7.5 Hz); 2.07–2.30 (m, 2H, ─CH₂─CH₃at C₁); 2.83–2.95 (m, 4H,

─CH₂─CH₃at C₈and ─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,

─CH₂at 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. R_f: 0.14 (M₃). FT-IR

(v_max, 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─CH₃at C₁, J = 7.5 Hz,

J = 7.2 Hz); 1.41 (t, 3H, ─CH₂─CH₃at C₈, J = 7.5 Hz, J = 7.2 Hz); 2.10–

2.30 (m, 2H, ─CH₂─CH₃at C₁); 2.65–2.98 (m, 4H, ─CH₂─CH₃at C₈

and CH₂─ at C₁); 3.09, 3.12 (d, 2H, ─CH₂─ at C₄); 3.68–3.93 (m, 4H,

─CH₂at 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. R_f: 0.25 (M₃). FT-IR

(v_max, 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─CH₃at 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─CH₃at C₈,

J = 7.5 Hz, J = 7.8 Hz); 2.10–2.30 (m, 2H, ─CH₂─CH₃at C₁); 2.87–2.95

(m, 4H, ─CH₂─CH₃at C₈and ─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,

─CH₂at 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. R_f: 0.34 (M₁). FT-IR (v_max, 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─CH₃at C₁, J = 7.2 Hz); 1.40 (t, 3H, ─CH₂─CH₃at C₈, J = 7.5 Hz,

J = 7.8 Hz); 2.01–2.31 (m, 2H, ─CH₂─CH₃at C₁); 2.59–3.15

(m, 6H, ─CH₂─CH₃at C₈and ─CH₂at C₁and ─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.

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3.61–3.99 (m, 2H, ─CH₂at 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. R_f: 0.49 (M₁). FT-IR (v_max, 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─CH₃at 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─CH₃at C₈J = 7.5 Hz); 2.04–

2.28 (m, 2H, ─CH₂─CH₃at C₁); 2.30 (s, 3H, Ar─CH₃); 2.62–3.14 (m,

6H, ─CH₂─CH₃, at C₈and ─CH₂at C₁and ─CH₂─ at C₄); 3.63–3.93

(m, 2H, ─CH₂at 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. R_f: 0.37 (M₁). FT-IR

(v_max, 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─CH₃at C₁, J = 7.2 Hz,

J = 7.5 Hz); 1.40 (t, 3H, ─CH₂─CH₃at C₈, J = 7.2 Hz, J = 7.8 Hz); 2.02–

2.31 (m, 2H, ─CH₂─CH₃at C₁); 2.76–3.14 (m, 6H, ─CH₂─CH₃at C₈and

─CH₂at C₁and ─CH₂─at C₄); 3.59–3.95 (m, 2H, ─CH₂at 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. R_f: 0.23 (M₁). FT-IR

(v_max, 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─CH₃at

C₁J = 7.2 Hz); 1.39 (t, 3H, ─CH₂─CH₃at C₈J = 7.5 Hz); 2.05–2.33 (m,

2H, ─CH₂─CH₃at C₁); 2.75–3.27 (m, 6H, ─CH₂─CH₃, at C₈and ─CH₂

at C₁and ─CH₂─ at C₄); 3.61–3.93 (m, 2H, ─CH₂at 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. R_f:0.71(M₃). FT-IR (v_max,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─CH₃at 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─CH₃at C₈, J = 7.5 Hz); 2.07–2.28 (m, 2H, ─CH₂─CH₃at C₁);

2.65–3.19 (m, 6H, ─CH₂─CH₃at C₈, ─CH₂─ at C₁and OH─CH₂─CH₃);

3.67–3.94 (m, 4H, ─CH₂─ at C₄and ─CH₂at 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

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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% CO₂in air. To determine the cell viability

cells were plated onto 96-well plates (1 × 10⁴cells/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% CO₂in 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 × 10⁴cells/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 × 10⁵cells/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.

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ORCID

[16] G. Tu, Y. Yan, X. Chen, Q. Lv, J Wang, S. Li, Drug Discov. Ther. 2013,

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Ş. Güniz Küçükgüzel

http://orcid.org/0000-0001-9405-8905

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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

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https://doi.org/10.1002/ardp.201700195

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

DOI: 10.1002/ardp.201700195

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