Design, synthesis and biological evaluation of a novel series of indole-3-carboxamide derivatives for cancer treatment as EGFR inhibitors

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Letters in Drug Design & Discovery, 2018, 15, 70-83

70

RESEARCH ARTICLE

ISSN: 1570-1808

eISSN: 1875-628X

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Design, Synthesis and Biological Evaluation of a Novel Series of Indole-3-

Carboxamide Derivatives for Cancer Treatment as EGFR Inhibitors

BENTHAM

S

CIENCE

Lan Zhang^a, Xin-Shan Deng^a, Guang-Peng Meng^a, Chao Zhang^a, Cong-Chong Liu^a, Gu-Zhou Chen^a,

Xu-Liang Jiang^a,*, Qing-Chun Zhao^a,band Chun Hu^a,*

^aKey Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical

b

University, Shenyang 110016, P.R. China; Department of Pharmacy, General Hospital of Shenyang Military Area

Command, Shenyang 110016, P.R. China

Abstract: Background: As reported EGFR is a sialoglycoprotein with tyrosine kinase activity

involved in control of cellular survival, multiplication, differentiation and metastasis. Dysregulation

or aberrant expression of EGFR has been implicated in cell transformation and having oncogenic

roles in a number of human cancers. Therefore EGFR has become a significant target for develop-

ing targeted therapy for cancer.

Methods: A novel series of indole-3-carboxamide derivatives as EGFR inhibitors were designed,

synthesized and evaluated for the anticancer activity in vitro against three EGFR high-expressed

A R T I C L E H I S T O R Yꢀ

cancer cell lines (A549, HeLa, and SW480), one EGFR low-expressed cell line (HepG2) and one

human liver normal cell line (HL7702) by MTT assay.

Received: February 07, 2017

Revised: September 07, 2017

Accepted: September 13, 2017

Results: The target compounds 6c, 6f, 6i, 6j, 6l, 6r, 6u and 6x exhibited potent anticancer activities

against the three tested cancer cell lines and weak cytotoxic effects on HepG2, which imply that the

DOI:

10.2174/1570180814666170929093258

target compounds are probably effective in inhibiting EGFR. And they also did not show measur-

able activities in HL7702, which imply the target compounds are likely to overcome the nonspecific

toxicity against normal cells. Particularly, the target compound 6x indicated equal to the positive

control erlotinib. In addition, molecular docking studies demonstrated the target compound 6x may

be the potential inhibitor to EGFR.

Conclusion: A new EGFR inhibitor scaffold and a preliminary discussion on their SARs provide

promising opportunities to guide further research on indole-3-carboxamide derivatives as novel

anticancer agents.

Keywords: Synthesis, indole-3-carboxamide derivatives, EGFR inhibitors, anticancer activity, structure-activity relationship,

cancer.

1. INTRODUCTION

oncogenic roles in a number of human cancers as lung,

breast, colon, gastric, prostatic, and head and neck cancers

[8, 9]. Therefore EGFR has become a significant target for

developing targeted therapy for cancer.

The World Cancer Report 2014 reveals that cancer is

becoming a primary cause of worldwide death, and in the

next two decades, cancer related deaths are likely to go up by

almost 70% [1]. Hence the research of novel cancer targets

and associated pathways establishes the era of molecular-

targeted medications [2-4]. Among the several target recep-

tors of cancer, the epidermal growth factor receptor (EGFR)

is a sialoglycoprotein with tyrosine kinase activity involved

in control of cellular survival, multiplication, differentiation

and metastasis [5-7]. Dysregulation or aberrant expression of

EGFR has been implicated in cell transformation and having

EGFR tyrosine kinase inhibitors (TKIs) show excellent

anticancer activity in patients and first generation EGFR

TKIs (gefitinib and erlotinib, Fig. 1) and the second genera-

tion EGFR TKIs (afatinib and lapatinib, Fig. 1) are approved

on a worldwide scale [10]. However, it has since been rec-

ognized that there are clinical problems in a subgroup of

patients who have primary and acquired resistance to the

EGFR TKIs [11-14]. As a consequence, there is an emergent

need to develop novel effective EGFR inhibitors that en-

hance the efficacy and safety for cancer treating.

*Address correspondence to these authors at the Key Laboratory of Structure-

based Drug Design & Discovery, Ministry of Education, Shenyang Pharma-

ceutical University, Shenyang 110016, P.R. China; Tel: + 86-24-43520246;

E-mails: xuliangjiang1975@163.com and chunhu@syphu.edu.cn

Indole is a key structural moiety known to have broad

biological activities. Previous studies by Hollis Showalter

have showed that a series of 4-anilinopyrimidoindole deriva-

1875-628X/18 $58.00+.00

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 71

ꢀ

Fig. (1). Chemical structures of gefitinib, erlotinib, afatinib and lapatinib.

_{Fig. (2). Design of a novel series of indole-3-carboxamide derivatives}ꢀ_{based on the biologically active compounds}

tives especially representative compound N-(3-bromophenyl)-

6-methoxy-9H-pyrimido[4,5-b]indol-4-amine (Fig. 2) exhibited

antiproliferative activity as potent inhibitors of EGFR [15].

Based on these tricyclic systems, the structure of lead com-

pound was simplified and the pyrimidine ring was opened to

get indole-3-carboxamide as the target compounds (Fig. 2).

With the purpose of improving anticancer activities, different

substituted benzylamine and various hydrophilic side chains

which have been frequently utilized in designing EGFR in-

hibitors were introduced into 3-position and 5-position of

indole-3-carboxamide scaffold, respectively. Moreover,

hereinafter docking studies were presented as well.

EGFR levels of A549, HeLa and SW480 cells were highly-

expressed [16-17], while that of HepG2 cells were low [18].

As expected, some target compounds exhibited obvious anti-

cancer activities and potency superior to positive control

drug against the tested cancer cells that expressed high level

of EGFR and did not show measurable activities in the very

low level of EGFR cells and human liver normal cells, which

implies that it is in all probability that the target compounds

are effective in inhibiting EGFR and conquering the toxicity

against normal cells. The structure–activity relationships

(SARs) were preliminary investigated based on the obtained

experimental results.

The synthesis of target compounds is described in

Scheme 1 with the intention of identifying much more con-

tributing anticancer drugs. Herein, the anticancer activity of

all target compounds was evaluated against A549 (human

lung adenocarcinoma), HeLa (Henrietta Lacks strain of can-

cer), SW480 (human colorectal cancer), HepG2 (human liver

cancer) and HL7702 (human liver) cell lines. As known, the

2. MATERIALS AND METHODS

2.1. Synthesis

All the starting materials were commercially available

and directly used without further purification. All the reac-

tion progress was monitored by thin layer chromatography

72 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

analysis, carried out on the silica gel plates (Qingdao Jiyida

silica reagent factory, Qingdao, China). All compounds'

melting points were determined on a melting-point apparatus

with microscope (Zhengzhou Mingyi Instrument Equipment

Corporation, Zhengzhou, China), and were uncorrected. ESI

mass spectra were performed on a Waters Q-TOF spec-

trometer (Waters Corporation, USA). The IR spectra were

recorded on a Bruker IFS55 spectrometer (KBr pellets

method). ¹H NMR and ¹³C NMR spectra were recorded on a

Bruker 400MHz or 101 MHz NMR spectrometer (Faellan-

den, Switzerland) with TMS as an internal standard and

DMSO-d₆or CDCl₃as the solvent, chemical shifts (d values)

and coupling constants (J values) are respectively given in ppm

and Hz. High-resolution mass spectral (HRMS) analyses

were recorded on Agilent Technologies 6530 Accurate-Mass

Q-TOF Mass Spectrometer (Agilent Technologies, USA).

temperature, the reaction was acidized with hydrochloric

acid to pH 2. Then the reaction mixture was filtered and the

residue was purified by recrystallization with ethanol to af-

ford the product as solid.

2.1.3.1.

Acid (4a)

5-Hydroxy-1,2-dimethyl-1H-indole-3-carboxylic

Yellow solid; yield: 77.0%; M.p.:216-217°C; ESI-MS,

m/z: calcd. 205.07(M⁺); found 204.1([M-H]^-); ¹H NMR (400

MHz, DMSO-d₆): δ 11.76 (s, 1H), 8.84 (s, 1H), 7.38 (d, J =

2.3 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 6.63 (dd, J = 8.7, 2.4

Hz, 1H), 3.63 (s, 3H), 2.65 (s, 3H).

2.1.3.2. 1-Ethyl-5-hydroxy-2-methyl-1H-indole-3-carboxylic

Acid (4b)

Yellow solid; yield: 68.3%; M.p.: 221-223°C; ESI-MS,

m/z: calcd. 219.09(M⁺); found 218.1([M-H]^-); ¹H NMR (400

MHz, DMSO-d₆): δ 11.76 (s, 1H), 8.84 (s, 1H), 7.38 (d, J =

2.3 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 6.62 (dd, J = 8.7, 2.4

Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 2.66 (s, 3H), 1.21 (t, J =

7.1 Hz, 3H).

2.1.1. General Procedure for the Preparation of Ethyl (Z)-

3-(substituted amino)but-2-enoate

To ethyl acetoacetate (15.01 g, 0.12 mol), methylamine

or ethylamine (0.36 mol, aqueous solution) was added drop-

wise and the reaction mixture was stirred at ambient tem-

perature for 3 h. The organic phase was successively washed

with water and brine and dried over anhydrous magnesium

sulfate to give the corresponding product as a transparent

liquid. The resulting product was used directly in the follow-

ing step without further purification.

2.1.4. General Procedure for the Preparation of 1-Alkyl-

N-(substituent

carboxamide

benzyl)-5-hydroxy-2-methyl-1H-indole-3-

A solution of 4a or 4b (0.020 mol), benzylamine or 4-

fluorobenzylamine (0.060 mol), 1-(3-(dimethylamino)propyl)-

3-ethyl-carbodiimide hydrochloride (EDCI, 5.75g, 0.030

mol), 1-hydroxybenzotriazole (HOBt, 4.05g, 0.030 mol) and

triethylamine (6.07 g, 0.060 mol) in N,N-dimethylformamide

(DMF, 100 mL) and dichloromethane (100 mL) was stirred

at ambient temperature for 24 h. Water (200 mL) was added

and the reaction mixture was stirred for 1 h. Then the mix-

ture was filtered and the residue was purified by recrystalli-

zation with 70% ethanol aqueous solution to afford product

as solid.

2.1.1.1. Ethyl (Z)-3-(methylamino)but-2-enoate (2a)

Yellow liquid; yield: 91.4%.

2.1.1.2. Ethyl (Z)-3-(ethylamino)but-2-enoate (2b)

White liquid; yield: 90.3%.

2.1.2. General Procedure for the Preparation of Ethyl 1-

alkyl-5-hydroxy-2-methyl-1H-indole-3-carboxylate

2.1.4.1.

N-Benzyl-5-hydroxy-1,2-dimethyl-1H-indole-3-

To a solution of benzoquinone (12.40 g, 0.11 mol) in ace-

tone (120 mL), 2a or 2b (0.11 mol) was added dropwise at

30°C, and the reaction mixture was stirred at this temperature

for 2 h. After cooling to room temperature, the reaction mix-

ture was concentrated in vacuo and the residue was purified by

recrystallization with acetone to afford the product as solid.

carboxamide (5a)

White solid; yield: 53.5%; M.p.: 211-213°C; ESI-MS,

m/z: calcd. 294.14(M⁺); found 295.1([M+H]⁺), 317.1

([M+Na]⁺).; ¹H NMR (400 MHz, DMSO-d₆): δ 8.81 (s, 1H),

7.94 (d, J = 2.3 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.36 – 7.22

(m, 5H), 7.16 (dd, J = 8.7, 2.3 Hz, 1H), 4.47 (d, J = 6.0 Hz,

2H), 3.61 (s, 3H), 2.56 (s, 3H).

2.1.2.1. Ethyl 5-hydroxy-1,2-dimethyl-1H-indole-3-carboxylate

(3a)

2.1.4.2.

N-(4-Fluorobenzyl)-5-hydroxy-1,2-dimethyl-1H-

White solid; yield: 75.2%; M.p.:208-210°C (lit.[19]: 208-

209°C). ESI-MS, m/z: calcd. 233.11(M⁺); found 234.1

([M+H]⁺), 356.1([M+Na]⁺).

indole-3-carboxamide (5b)

White solid; yield: 73.0%; M.p.: 217-218°C; ESI-MS,

m/z: calcd. 312.13(M⁺); found 313.1([M+H]⁺), 335.1

([M+Na]⁺).; ¹H NMR (400 MHz, DMSO-d₆): δ 8.82 (s, 1H),

7.95 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.4, 5.7 Hz, 2H), 7.24

(d, J = 8.7 Hz, 1H), 7.18 – 7.11 (m, 3H), 4.44 (d, J = 6.0 Hz,

2H), 3.62 (s, 3H), 2.56 (s, 3H).

2.1.2.2. Ethyl 1-ethyl-5-hydroxy-2-methyl-1H-indole-3-

carboxylate (3b)

White solid; yield: 76.8%; M.p.:184-186°C (lit.[20]: 184-

185°C). ESI-MS, m/z: calcd. 247.12(M⁺); found 248.1

([M+H]⁺), 270.1([M+Na]⁺).

2.1.4.3. N-Benzyl-1-ethyl-5-hydroxy-2-methyl-1H-indole-3-

carboxamide (5c)

2.1.3. General Procedure for the Preparation of 1-Alkyl -5-

hydroxy-2-methyl-1H-indole-3-carboxylic Acid

Yellow solid; yield: 59.2%; M.p.: 189-190°C; ESI-MS,

m/z: calcd. 308.15(M+); found 309.2([M+H]+); 1H NMR

(400 MHz, DMSO-d6): δ 8.81 (s, 1H), 7.94 (d, J = 2.3 Hz,

1H), 7.35 (d, J = 8.7 Hz, 1H), 7.34 – 7.23 (m, 5H), 7.15 (dd,

A solution of 3a or 3b (0.0043 mol) and sodium hydrox-

ide (17.15 g, 0.043 mol) in ethanol (100 mL) and water (50

mL) was stirred at reflux for 12 h. After cooling to ambient

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 73

J = 8.7, 2.3 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H), 4.11 (q, J = 7.1

Hz, 2H), 2.56 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H).

160.74(C₈), 153.88(C₅), 139.34(C₂), 139.29(C-Ph), 128.83

(C₉), 127.93(CH-Ph), 127.42(CH-Ph), 125.76(CH-Ph), 111.12

(C₆), 110.31(C₃), 107.52(C₇), 103.55(C₄), 68.65(C_1’’), 49.10

(NCH₂), 47.63(NCH₂), 43.60(C_2’), 29.75(NCH₃), 22.30

(CH₂), 20.80(CH₂), 11.91(indole-CH₃), 11.49(CH₃), 11.39

(CH₃); HRMS (ESI): calcd for C₂₆H₃₃N₃O₃, [M]⁺m/z,

435.2522; found, 436.2581([M+H]⁺), 458.2391([M+Na]⁺).

2.1.4.4.

1-Ethyl-N-(4-fluorobenzyl)-5-hydroxy-2-methyl-

1H-indole-3-carboxamide (5d)

Yellow solid; yield: 58.4%; M.p.: 167-168°C; ESI-MS,

m/z: calcd. 326.14(M⁺); found 327.1([M+H]⁺), 349.1

([M+Na]⁺).; ¹H NMR (400 MHz, DMSO-d₆): δ 8.81 (s, 1H),

7.94 (d, J = 2.3 Hz, 1H), 7.28 (dd, J = 8.5, 5.6 Hz, 2H), 7.16

(d, J = 8.7 Hz, 1H), 7.11 – 6.96 (m, 3H), 4.56 (d, J = 5.8 Hz,

2H), 4.07 (q, J = 7.2 Hz, 2H), 2.67 (s, 3H), 1.30 (t, J = 7.2

Hz, 3H).

2.1.5.3. N-Benzyl-5-[2-(di-sec-butylamino)-2-oxoethoxy]-

1,2-dimethyl-1H-indole-3-carboxamide (6c)

Yellow solid; yield: 70.9%; M.p.: 105-106°C; IR: (KBr,

cm^-1): υ 3425.2, 2919.0, 2850.0, 1621.9, 1485.5, 1384.5,

1194.7, 1120.5, 821.2, 778.6, 697.9; ESI-MS, m/z: calcd.

463.28(M⁺); found 464.3([M+H]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.43 (d, J = 2.3 Hz, 1H, indole-H₇), 7.38 – 7.27

(m, 5H, Ar–H), 7.19 (d, J = 8.9 Hz, 1H, indole-H₄), 6.90 (dd,

J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.71 (d, J = 5.6 Hz, 2H,

NHCH₂), 4.70 (s, 2H, OCH₂), 3.65 (s, 3H, NCH₃), 3.20 –

3.11(m, 2H, NCH), 2.72 (s, 3H, indole-CH₃), 1.29 – 1.25 (m,

4H, CH₂), 0.92 (d, J = 6.7 Hz, 6H, CH₃), 0.78 (t, J = 6.7 Hz,

6H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 168.71(C_1’),

166.30(C_2’’), 160.17(C₈), 153.82(C₅), 139.28(C₂), 137.48(C-

Ph), 128.83(C₉), 127.94(CH-Ph), 127.42(CH-Ph), 125.74

(CH-Ph), 111.09(C₆), 110.26(C₃), 107.49(C₇), 103.73(C₄),

68.60(C_1’’), 54.48(NCH), 52.64(NCH), 43.68(C_2’), 29.84

(NCH₃), 27.46(CH₂), 26.26(CH₂), 20.25(CH₃), 20.19(CH₃),

11.91(indole-CH₃), 11.00(CH₃), 10.54(CH₃); HRMS (ESI):

calcd for C₂₈H₃₇N₃O₃, [M]⁺m/z, 463.2835; found, 464.

2892([M+H]⁺), 486.2705([M+Na]⁺).

2.1.5. General Procedure for the Preparation of Indole-3-

carboxamide Derivatives 6a-6x

A solution of 5a, 5b, 5c or 5d (0.0034 mol), 2-

chloroacetamide(0.017 mol), anhydrous potassium carbon-

ate(4.70 g, 0.034 mol), potassium iodide(0.06 g, 0.00034

mol) in N,N-dimethylformamide (DMF, 20 mL) was stirred

at 80^oC for 16 h. Water (100 mL) was added and the reaction

mixture was stirred for 4 h. The mixture was filtered and the

residue was purified by column chromatography on silica gel

using petroleum ether/ ethyl acetate(5/1, v/v) as eluent to

afford the corresponding products (6a-6x).

2.1.5.1.

N-Benzyl-5-[2-(diethylamino)-2-oxoethoxy]-1,2-

dimethyl-1H-indole-3-carboxamide (6a)

White solid; yield: 63.0%; M.p.: 119-120°C; IR: (KBr,

cm^-1): υ 3444.3, 2927.6, 1660.6, 1620.2, 1431.2, 1383.6,

1257.3, 1190.8, 1085.3, 834.3, 776.2, 745.1, 701.8; ESI-MS,

m/z: calcd. 407.22(M⁺); found 408.3([M+H]⁺), 430.2

2.1.5.4.

N-Benzyl-1,2-dimethyl-5-[2-oxo-2-(piperidin-1-

yl)ethoxy]-1H-indole-3-carboxamide (6d)

1

([M+Na]⁺), 446.2([M+K]⁺).; H NMR (400 MHz, CDCl₃): δ

White solid; yield: 58.5%; M.p.: 78-80°C; IR: (KBr, cm^-1):

υ 3429.6, 2923.2, 2853.6, 1641.2, 1618.1, 1488.8, 1440.1,

1212.0, 1163.4, 1082.8, 1017.8, 874.0, 699.6; ESI-MS, m/z:

calcd. 419.22(M⁺); found 420.4([M+H]⁺), 442.3([M+Na]⁺);

¹H NMR (400 MHz, CDCl₃): δ 7.43 (d, J = 2.4 Hz, 1H, in-

dole-H₇), 7.38 – 7.26 (m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H,

indole-H₄), 6.91 (dd, J = 8.9, 2.4 Hz, 1H, indole-H₆), 4.71 (d,

J = 5.8 Hz, 2H, NHCH₂), 4.66 (s, 2H, OCH₂), 3.66 (s, 3H,

NCH₃), 3.52 (t, J = 5.3 Hz, 2H, NCH₂), 3.45 (t, J = 5.0 Hz,

2H, NCH₂), 2.72 (s, 3H, indole-CH₃), 1.61-1.53 (m, 6H,

CH₂); ¹³C NMR (101 MHz, CDCl₃): δ 166.52(C_1’),

166.15(C_2’’), 160.43(C₈), 153.62(C₅), 142.55(C₂), 139.16(C-

Ph), 128.69(C₉), 127.83(CH-Ph), 127.29(CH-Ph), 125.64

(CH-Ph), 111.03(C₆), 110.24(C₃), 107.42(C₇), 103.31(C₄),

68.65(C_1’’), 46.44(C_2’), 43.56(NCH₂), 43.17(NCH₂), 29.62

(NCH₃), 26.49(CH₂), 25.55(CH₂), 24.48(CH₂), 11.78(indole-

CH₃); HRMS (ESI): calcd for C₂₅H₂₉N₃O₃, [M]⁺m/z,

419.2209; found, 442.2077([M+Na]⁺).

7.43 (d, J = 2.3 Hz, 1H, indole-H₇), 7.37 – 7.27 (m, 5H, Ar–

H), 7.21 (d, J = 8.9 Hz, 1H, indole-H₄), 6.91 (dd, J = 8.9, 2.4

Hz, 1H, indole-H₆), 4.71 (d, J = 5.7 Hz, 2H, NHCH₂), 4.66

(s, 2H, OCH₂), 3.66 (s, 3H, NCH₃), 3.38 (dd, J = 7.1, 2.9 Hz,

2H, NCH₂), 3.35 (dd, J = 7.1, 2.9 Hz, 2H, NCH₂), 2.72 (s,

3H, indole-CH₃), 1.17 (t, J = 7.1 Hz, 3H, CH₃), 1.11 (t, J =

7.1 Hz, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ

167.51(C_1’), 166.30(C_2’’), 158.24(C₈), 153.86(C₅), 139.28(C₂,

C-Ph), 132.28(CH-Ph), 128.83(C₉), 127.97(CH-Ph), 127.43

(CH-Ph), 126.95(CH-Ph), 111.24(C₆), 110.33(C₃), 107.55

(C₇), 103.50(C₄), 68.71(C_1’’), 43.69(C_2’), 41.71(NCH₂), 40.39

(NCH₂), 29.76(NCH₃), 12.97(CH₃), 12.78(CH₃), 11.92

(indole-CH₃); HRMS (ESI): calcd for C₂₄H₂₉N₃O₃, [M]⁺m/z,

407.2209; found, 408.2270([M+H]⁺), 430.2083([M+Na]⁺).

2.1.5.2. N-Benzyl-5-[2-(dipropylamino)-2-oxoethoxy]-1,2-

dimethyl-1H-indole-3-carboxamide (6b)

White solid; yield: 73.0%; M.p.: 123-125°C; IR: (KBr,

cm^-1): υ 3349.0, 2964.9, 2871.1, 1656.2, 1484.3, 1382.6,

1270.8, 1190.0, 1143.3, 1084.6, 926.2, 839.0, 798.2, 741.2,

697.8; ESI-MS, m/z: calcd. 435.25(M⁺); found 436.2

2.1.5.5.

N-Benzyl-1,2-dimethyl-5-[2-(morpholin-4-yl)-2-

oxoethoxy]-1H-indole-3-carboxamide (6e)

1

([M+H]⁺), 458.2([M+Na]⁺); H NMR (400 MHz, CDCl₃): δ

White solid; yield: 54.6%; M.p.: 110-112°C; IR: (KBr,

cm^-1): υ 3442.6, 2856.0’ 1653.8, 1483.8, 1384.1, 1274.2,

1207.7, 1162.8, 1031.3, 923.2, 850.8, 698.4; ESI-MS, m/z:

calcd. 421.20(M⁺); found 422.2([M+H]⁺), 444.2([M+Na]⁺);

¹H NMR (400 MHz, CDCl₃): δ 7.44 (d, J = 2.3 Hz, 1H, in-

dole-H₇), 7.38 – 7.27 (m, 5H, Ar–H), 7.21 (d, J = 8.9 Hz, 1H,

indole-H₄), 6.89 (dd, J = 8.9, 2.4 Hz, 1H, indole-H₆), 4.71 (d,

J = 5.7 Hz, 2H, NHCH₂), 4.68 (s, 2H, OCH₂), 3.67 (s, 3H,

7.43 (d, J = 2.3 Hz, 1H, indole-H₇), 7.38 – 7.26 (m, 5H, Ar–

H), 7.20 (d, J = 8.9 Hz, 1H, indole-H₄), 6.90 (dd, J = 8.9, 2.4

Hz, 1H, indole-H₆), 4.71 (d, J = 5.7 Hz, 2H, NHCH₂), 4.67

(s, 2H, OCH₂), 3.66 (s, 3H, NCH₃), 3.26 (q, J = 7.0 Hz, 4H,

NCH₂), 2.72 (s, 3H, indole-CH₃), 1.56 – 1.48 (m, 4H, CH₂),

0.89 (t, J = 7.4 Hz, 3H, CH₃), 0.84 (t, J = 7.4 Hz, 3H, CH₃);

¹³C NMR (101 MHz, CDCl₃): δ 167.96(C_1’), 166.32(C_2’’),

74 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

NCH₃), 3.63 – 3.57 (m, 8H, CH₂), 2.72 (s, 3H, indole-CH₃);

¹³C NMR (101 MHz, CDCl₃): δ 167.08(C_1’), 166.20(C_2’’),

160.23(C₈), 153.39(C₅), 142.69(C₂), 139.25(C-Ph), 128.86

(C₉), 127.99(CH-Ph), 127.49(CH-Ph), 125.85(CH-Ph),

111.08(C₆), 110.46(C₃), 107.64(C₇), 103.35(C₄), 68.67(C_1’’),

66.95(OCH₂), 66.91(OCH₂), 46.07(C_2’), 43.73(NCH₂), 42.53

(NCH₂), 29.79(NCH₃), 11.94(indole-CH₃); HRMS (ESI):

calcd for C₂₄H₂₇N₃O₄, [M]⁺m/z, 421.2002; found, 422.

2065([M+H]⁺), 444.1875([M+Na]⁺).

NMR (400 MHz, CDCl₃): δ 7.40 (dd, J = 8.7, 5.4 Hz, 2H,

Ar–H), 7.28 (d, J = 2.3 Hz, 1H, indole-H₇), 7.21 (d, J = 8.9

Hz, 1H, indole-H₄), 7.03 (t, J = 8.7 Hz, 2H, Ar–H), 6.89 (dd,

J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.70 (s, 2H, OCH₂), 4.67 (d,

J = 5.6 Hz, 2H, NHCH₂), 3.66 (s, 3H, NCH₃), 3.29 – 3.24

(m, 4H, NCH₂), 2.72 (s, 3H, indole-CH₃), 1.54 – 1.47 (m,

4H, CH₂), 0.90 (t, J = 7.4 Hz, 3H, CH₃), 0.83 (t, J = 7.4 Hz,

3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 167.42(C_1’),

166.29(C_2’’), 160.95(C₈), 160.63(C-F), 154.41(C₅), 138.45

(C₂), 133.69(C-C₆H₄F), 129.65(CH-C₆H₄F), 129.57(CH-

C₆H₄F), 128.06(C₉), 115.70(CH-C₆H₄F), 115.55(CH-C₆H₄F),

111.05(C₆), 110.87(C₃), 110.36(C₇), 103.47(C₄), 68.75(C_1’’),

58.41(NCH₂), 57.95(NCH₂), 49.11(C_2’), 29.76(NCH₃), 22.30

(CH₂), 20.80(CH₂), 12.15(indole-CH₃), 11.47(CH₃), 11.35

(CH₃); HRMS (ESI): calcd for C₂₆H₃₂FN₃O₃, [M]⁺m/z,

453.2428; found, 477.2335([M+Na]⁺).

2.1.5.6. N-Benzyl-5-{2-{[2-(2-methoxyphenoxy)ethyl]amino}-

2-oxoethoxy}-1,2-dimethyl-1H-indole-3-carboxamide (6f)

White solid; yield: 58.8%; M.p.: 180-181°C; IR: (KBr,

cm^-1): υ 3426.8, 2923.0, 2852.3, 1628.8, 1384.4, 1253.4,

1124.5, 777.8, 618.2; ESI-MS, m/z: calcd. 501.23(M⁺);

found 502.4([M+H]⁺), 524.4([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.40 (d, J = 2.3 Hz, 1H, indole-H₇), 7.38 – 7.27

(m, 5H, benzyl-H), 7.22 (d, J = 8.9 Hz, 1H, indole-H₄), 6.90

– 6.81 (m, 5H, Ar–H), 4.68 (d, J = 5.6 Hz, 2H, NHCH₂),

4.50 (s, 2H, OCH₂CO), 4.10 (t, J = 5.1 Hz, 2H, OCH₂), 3.76

(s, 3H, NCH₃), 3.74 – 3.71 (m, 2H, CH₂), 3.66 (s, 3H,

OCH₃), 2.72 (s, 3H, indole-CH₃); ¹³C NMR (101 MHz,

CDCl₃): δ 169.00(C_1’), 168.96(C_2’’), 160.50(C₈), 153.10(C₅),

150.28(O-C-Ph), 150.22(O-C-Ph), 139.13(C₂), 137.31(C-

Ph), 128.95(C₉), 127.87(CH-Ph), 127.60(CH-Ph), 125.88

(CH-Ph), 122.48(CH-Ph), 121.15(CH-Ph), 120.30(CH-Ph),

115.62(CH-Ph), 112.19(C₆), 111.07(C₃), 110.46(C₇), 103.83

(C₄), 69.01(OCH₂), 68.84(C_1’’), 56.14(OCH₃), 43.72(C_2’),

38.50(NHCH₂), 29.79(NCH₃), 11.91(indole-CH₃); HRMS

(ESI): calcd for C₂₉H₃₁N₃O₅, [M]⁺m/z, 501.2264; found,

502.2322([M+H]⁺), 524.2134([M+Na]⁺).

2.1.5.9.

5-[2-(Di-sec-butylamino)-2-oxoethoxy]-N-(4-

fluorobenzyl)-1,2-dimethyl-1H-indole-3-carboxamide (6i)

White solid; yield: 57.1%; M.p.: 137-139°C; IR: (KBr,

cm^-1): υ 3355.2, 2961.8, 1656.6, 1540.3, 1485.8, 1385.1,

1215.7, 1145.0, 1082.5, 821.1, 795.3, 758.4, 712.4, 589.9;

ESI-MS, m/z: calcd. 481.27(M⁺); found 482.4([M+H]⁺),

1

504.4([M+Na]⁺); H NMR (400 MHz, CDCl₃): δ 7.41 (dd, J

= 8.7, 5.4 Hz, 2H, Ar–H), 7.30 (d, J = 2.3 Hz, 1H, indole-

H₇), 7.20 (d, J = 8.9 Hz, 1H, indole-H₄), 7.02 (t, J = 8.7 Hz,

2H, Ar–H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.73 (s,

2H, OCH₂), 4.67 (d, J = 5.6 Hz, 2H, NHCH₂), 3.66 (s, 3H,

NCH₃), 3.20 – 3.13 (m, 2H, NCH), 2.72 (s, 3H, indole-CH₃),

2.01 – 1.87 (m, 4H, CH₂), 0.93 (d, J = 6.7 Hz, 6H, CH₃),

0.77 (t, J = 6.7 Hz, 6H, CH₃); ¹³C NMR (101 MHz, CDCl₃):

δ 168.79(C_1’), 166.29(C_2’’), 160.65(C₈), 160.04(C-F), 153.84

(C₅), 139.85(C₂), 132.31(C-C₆H₄F), 129.65(CH-C₆H₄F), 129.

57(CH-C₆H₄F), 127.69(C₉), 115.71(CH-C₆H₄F), 115.50(CH-

C₆H₄F), 111.03(C₆), 110.31(C₃), 107.29(C₇), 103.77(C₄),

68.69(C_1’’), 54.46(NCH), 54.28(NCH), 52.62(C_2’), 29.76

(NCH₃), 27.44(CH₂), 26.25(CH₂), 18.87(CH₃), 18.81(CH₃),

11.92(indole-CH₃), 10.07(CH₃), 9.83(CH₃); HRMS (ESI):

calcd for C₂₈H₃₆FN₃O₃, [M]⁺m/z, 481.2741; found,

482.2804([M+H]⁺), 504.2614([M+Na]⁺).

2.1.5.7. 5-[2-(Diethylamino)-2-oxoethoxy]-N-(4-fluorobenzyl)-

1,2-dimethyl-1H-indole-3-carboxamide (6g)

White solid; yield: 50.4%; M.p.: 133-134°C; IR: (KBr,

cm^-1): υ 3442.8, 2972.6, 2931.4, 1658.0, 1619.9, 1487.1,

1383.6, 1218.2, 1143.2, 1086.9, 974.6, 822.1, 757.6, 568.6;

ESI-MS, m/z: calcd. 425.21(M⁺); found 426.3([M+H]⁺),

1

448.4([M+Na]⁺); H NMR (400 MHz, CDCl₃): δ 7.41 (dd, J

= 8.6, 5.4 Hz, 2H, Ar–H), 7.29 (d, J = 2.3 Hz, 1H, indole-

H₇), 7.21 (d, J = 8.9 Hz, 1H, indole-H₄), 7.03 (t, J = 8.7 Hz,

2H, Ar–H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.68 (s,

2H, OCH₂), 4.66 (d, J = 5.6 Hz, 2H, NHCH₂), 3.66 (s, 3H,

NCH₃), 3.37 (q, J = 6.9 Hz, 4H, NCH₂), 2.72 (s, 3H, indole-

CH₃), 1.17 (t, J = 7.1 Hz, 3H, CH₃), 1.10 (t, J = 7.1 Hz, 3H,

CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 167.60(C_1’), 166.29

(C_2’’), 161.77(C₈), 161.46(C-F), 153.89(C₅), 143.01(C₂),

132.28(C-C₆H₄F), 129.73(CH-C₆H₄F), 129.67(CH-C₆H₄F),

127.61(C₉), 115.70(CH-C₆H₄F), 115.48(CH-C₆H₄F), 111.19

(C₆), 110.38(C₃), 109.37(C₇), 103.52(C₄), 68.75(C_1’’), 41.72

(C_2’), 40.41(NCH₂), 40.33(NCH₂), 29.73(NCH₃), 14.46

(CH₃), 12.95(CH₃), 11.92(indole-CH₃); HRMS (ESI): calcd

for C₂₄H₂₈FN₃O₃, [M]⁺m/z, 425.2115; found, 426.2178

([M+H]⁺), 448.1989([M+Na]⁺).

2.1.5.10.

N-(4-Fluorobenzyl)-1,2-dimethyl-5-[2-oxo-2-

(piperidin-1-yl)ethoxy]-1H-indole-3-carboxamide (6j)

White solid; yield: 40.0%; M.p.: 92-94°C; IR: (KBr, cm^-1):

υ 3423.8, 2920.5, 2851.1, 1626.7, 1509.2, 1445.4, 1220.0,

1156.9, 1014.3, 874.3, 803.4, 714.5; ESI-MS, m/z: calcd.

437.21(M⁺); found 438.4([M+H]⁺), 560.4([M+Na]⁺); ¹H

NMR (400 MHz, CDCl₃): δ 7.41 (dd, J = 8.6, 5.4 Hz, 2H,

Ar–H), 7.29 (d, J = 2.4 Hz, 1H, indole-H₇), 7.21 (d, J = 8.9

Hz, 1H, indole-H₄), 7.03 (t, J = 8.7 Hz, 2H, Ar–H), 6.90 (dd,

J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.69 (s, 2H, OCH₂), 4.67 (d,

J = 5.8 Hz, 2H, NHCH₂), 3.66 (s, 3H, NCH₃), 3.51 (t, J = 5.2

Hz, 2H, NCH₂), 3.45 (t, J = 5.1 Hz, 2H, NCH₂), 2.72 (s, 3H,

indole-CH₃), 1.55 – 1.47 (m, 6H, CH₂); ¹³C NMR (101 MHz,

CDCl₃): δ 166.95(C_1’), 166.70(C_2’’), 159.27(C₈), 159.16(C-

F), 153.80(C₅), 136.18(C₂), 132.27(C-C₆H₄F), 129.69(CH-

C₆H₄F), 129.61(CH-C₆H₄F), 127.99(C₉), 115.70(CH-C₆H₄F),

115.49(CH-C₆H₄F), 111.15(C₆), 110.41(C₃), 108.77(C₇),

103.44(C₄), 68.83(C_1’’), 49.25(NCH₂), 49.14(NCH₂), 43.32

(C_2’), 29.77(NCH₃), 25.69(CH₂), 25.63(CH₂), 24.60(CH₂),

11.92(indole-CH₃); HRMS (ESI): calcd for C₂₅H₂₈FN₃O₃,

2.1.5.8. 5-[2-(Dipropylamino)-2-oxoethoxy]-N-(4-fluorobenzyl)-

1,2-dimethyl-1H-indole-3-carboxamide (6h)

White solid; yield: 73.1%; M.p.: 145-147°C; IR: (KBr,

cm^-1): υ 3446.5, 2930.0, 2360.9, 1655.5, 1508.8, 1484.3,

1383.4, 1271.9, 1190.1, 1095.7, 819.2, 798.4, 777.4; ESI-

MS, m/z: calcd. 453.24(M⁺); found 454.4([M+H]⁺); ¹H

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 75

[M]⁺m/z, 437.2115; found, 438.2178([M+H]⁺), 460.1988

([M+Na]⁺).

OCH₂), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH₂), 3.36 (q, J =

7.0 Hz, 4H, NCH₂), 2.72 (s, 3H, indole-CH₃), 1.33 (t, J = 7.2

Hz, 3H, CH₃), 1.17 (t, J = 7.1 Hz, 3H, CH₃), 1.11 (t, J = 7.1

Hz, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 167.53(C_1’),

166.34(C_2’’), 153.79(C₅), 141.82(C₂), 139.29(C-Ph), 128.81

(C₈), 128.72(CH-Ph), 128.59(CH-Ph), 127.96(C₉), 127.41

(CH-Ph), 126.28(CH-Ph), 126.01(CH-Ph), 111.23(C₆),

110.35(C₃), 107.65(C₇), 103.57(C₄), 68.70(C_1’’), 43.68(C_2’),

41.69(NCH₂), 40.38(NCH₂), 38.02(NCH₂), 15.10(CH₃),

14.47(CH₃), 12.96(CH₃), 11.63(indole-CH₃); HRMS (ESI):

calcd for C₂₅H₃₁N₃O₃, [M]⁺m/z, 421.2365; found, 422.

2427([M+H]⁺), 444.2234([M+Na]⁺).

2.1.5.11. N-(4-Fluorobenzyl)-1,2-dimethyl-5-[2-(morpholin-

4-yl)-2-oxoethoxy]-1H-indole-3-carboxamide (6k)

Yellow solid; yield: 75.9%; M.p.: 181-182°C; IR: (KBr,

cm^-1): υ 3422.9, 2919.7, 2860.2, 1677.1, 1627.6, 1506.2,

1444.1, 1214.8, 1162.4, 1117.0, 1032.2, 845.3, 808.7, 720.8;

ESI-MS, m/z: calcd. 439.19(M⁺); found 440.3([M+H]⁺),

1

462.3([M+Na]⁺); H NMR (400 MHz, CDCl₃): δ 7.41 (dd, J

= 8.5, 5.4 Hz, 2H, Ar–H), 7.28 (d, J = 2.4 Hz, 1H, indole-

H₇), 7.21 (d, J = 8.9 Hz, 1H, indole-H₄), 7.03 (t, J = 8.7 Hz,

2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.70 (s,

2H, OCH₂), 4.67 (d, J = 5.8 Hz, 2H, NHCH₂), 3.66 (s, 3H,

NCH₃), 3.63 – 3.58 (m, 8H, CH₂), 2.71 (s, 3H, indole-CH₃);

¹³C NMR (101 MHz, CDCl₃): δ 167.13(C_1’), 166.18(C_2’’),

163.42(C₈), 161.03(C-F), 153.42(C₅), 142.89(C₂), 132.35(C-

C₆H₄F), 129.69(CH-C₆H₄F), 129.61(CH-C₆H₄F), 125.77(C₉),

115.73(CH-C₆H₄F), 115.51(CH-C₆H₄F), 111.06(C₆), 110.51

(C₃), 107.49(C₇), 103.33(C₄), 68.73(C_1’’), 66.94(OCH₂), 66.90

(OCH₂), 46.07(C_2’), 42.96(NCH₂), 42.54(NCH₂), 29.79

(NCH₃), 11.93(indole-CH₃); HRMS (ESI): calcd for C₂₄H₂₆

FN₃O₄, [M]⁺m/z, 439.1907; found, 440.1971([M+H]⁺),

462.1779([M+Na]⁺).

2.1.5.14. N-Benzyl-5-[2-(dipropylamino)-2-oxoethoxy]-1-

ethyl-2-methyl-1H-indole-3-carboxamide (6n)

White solid; yield: 74.3%; M.p.: 129-130°C; IR: (KBr,

cm^-1): υ 3444.2, 2930.3, 1657.5, 1618.2, 1482.3, 1383.6,

1272.0, 1193.7, 1145.4, 1090.3, 795.4, 698.8; ESI-MS, m/z:

calcd. 449.27(M⁺); found 450.2([M+H]⁺), 472.3([M+Na]⁺);

¹H NMR (400 MHz, CDCl₃): δ 7.42 (d, J = 2.2 Hz, 1H, in-

dole-H₇), 7.37 – 7.27 (m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H,

indole-H₄), 6.89 (dd, J = 8.9, 2.3 Hz, 1H, indole-H₆), 4.70 (d,

J = 5.8 Hz, 2H, NHCH₂), 4.67 (s, 2H, OCH₂), 4.11 (q, J =

7.1 Hz, 2H, indole-NCH₂), 3.29 – 3.21 (m, 4H, NCH₂), 2.71

(s, 3H, indole-CH₃), 1.60 – 1.52 (m, 4H, CH₂), 1.34 (t, J =

7.3 Hz, 3H, CH₃), 0.89 (t, J = 7.4 Hz, 3H, CH₃), 0.84 (t, J =

7.4 Hz, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ

167.95(C_1’), 166.34(C_2’’), 153.79(C₅), 141.87(C₂), 139.30(C-

Ph), 128.90(C₈), 128.80(CH-Ph), 128.58(CH-Ph), 127.92

(C₉), 127.39(CH-Ph), 126.79(CH-Ph), 125.99(CH-Ph),

111.10(C₆), 110.32(C₃), 107.61(C₇), 103.62(C₄), 68.61(C_1’’),

49.07(C_2’), 47.61(NCH₂), 43.66(NCH₂), 38.00(NCH₂),

22.28(CH₂), 20.79(CH₂), 15.08(CH₃), 11.62(indole-CH₃),

11.47(CH₃), 11.38(CH₃); HRMS (ESI): calcd for C₂₇H₃₅

N₃O₃, [M]⁺m/z, 449.2678; found, 450.2740([M+H]⁺),

472.2550([M+Na]⁺).

2.1.5.12. N-(4-Fluorobenzyl)-5-{2-{[2-(2-methoxyphenoxy)

ethyl]amino}-2-oxoethoxy}-1,2-dimethyl-1H-indole-3-

carboxamide (6l)

White solid; yield: 43.4%; M.p.: 187-189°C; IR: (KBr,

cm^-1): υ 3453.5, 3238.6, 2912.8, 1681.0, 1627.1, 1506.3,

1485.9, 1384.4, 1252.9, 1222.8, 1126.3, 1026.5, 829,.6,

790.9, 736.4; ESI-MS, m/z: calcd. 519.22(M⁺); found

520.3([M+H]⁺), 542.2([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.35 (dd, J = 8.6, 5.4 Hz, 2H, 4-fluorobenzyl-H),

7.23 (d, J = 2.3 Hz, 1H, indole-H₇), 7.19 (d, J = 8.9 Hz, 1H,

indole-H₄), 7.03 (t, J = 8.7 Hz, 2H, 4-fluorobenzyl-H), 6.95

(dd, J = 3.8, 1.2 Hz, 1H, indole-H₆), 6.89 – 6.83 (m, 4H, Ar–

H), 4.64 (d, J = 5.8 Hz, 2H, NHCH₂), 4.53 (s, 2H, OCH₂CO),

4.10 (t, J = 5.1 Hz, 2H, OCH₂), 3.75 (s, 3H, NCH₃), 3.74 –

3.70 (m, 2H, CH₂), 3.66 (s, 3H, OCH₃), 2.71 (s, 3H, indole-

CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 169.67(C_1’), 167.83

(C_2’’), 160.94(C₈), 160.57(C-F), 153.28(C₅), 150.27(O-C-Ph),

150.21(O-C-Ph), 139.09(C₂), 133.42(C-C₆H₄F), 129.45(CH-

C₆H₄F), 129.26(CH-C₆H₄F), 127.58(C₉), 123.55(CH-Ph),

122.43(CH-Ph), 121.39(CH-Ph), 115.80(CH-C₆H₄F), 115.52

(CH-C₆H₄F), 112.20(C₆), 111.17(C₃), 110.36(C₇), 103.81

(C₄), 69.07(OCH₂), 68.73(C_1’’), 56.19(OCH₃), 43.62(C_2’),

40.21(NHCH₂), 29.97(NCH₃), 11.93(indole-CH₃); HRMS

(ESI): calcd for C₂₉H₃₀FN₃O₅, [M]⁺m/z, 519.2169; found,

520.2231([M+H]⁺), 542.2037([M+Na]⁺).

2.1.5.15. N-Benzyl-5-[2-(di-sec-butylamino)-2-oxoethoxy]-

1-ethyl-2-methyl-1H-indole-3-carboxamide (6o)

White solid; yield: 56.0%; M.p.: 101-103°C; IR: (KBr,

cm^-1): υ 3423.1, 2959.1, 2920.8, 1656.7, 1621.6, 1543.7,

1484.8, 1422.4, 1384.8, 1209.4, 1143.3, 1091.9, 817.5,

744.8, 698.8; ESI-MS, m/z: calcd. 477.30(M⁺); found

478.4([M+H]⁺), 500.3([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.43 (d, J = 2.3 Hz, 1H, indole-H₇), 7.38 – 7.25

(m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H, indole-H₄), 6.89 (dd,

J = 8.8, 2.3 Hz, 1H, indole-H₆), 4.71 (d, J = 5.8 Hz, 2H,

NHCH₂), 4.66 (s, 2H, OCH₂), 4.12 (q, J = 7.1 Hz, 2H, in-

dole-NCH₂), 3.21 – 3.14 (m, 2H, NCH), 2.72 (s, 3H, indole-

CH₃), 2.02 – 1.67 (m, 4H, CH₂), 1.33 (t, J = 7.1 Hz, 3H,

CH₃), 0.92(d, J = 6.7 Hz, 6H, CH₃), 0.79 (t, J = 6.7 Hz, 6H,

CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 168.70(C_1’), 166.35

(C_2’’), 153.73(C₅), 141.92(C₂), 139.29(C-Ph), 128.82(C₈),

128.55(CH-Ph), 128.28(CH-Ph), 127.94(C₉), 127.40(CH-Ph),

126.86(CH-Ph), 125.99(CH-Ph), 111.06(C₆), 110.28(C₃),

107.57(C₇), 103.82(C₄), 68.58(C_1’’), 54.47(NCH), 52.62

(NCH), 43.67(C_2’), 38.00(NCH₂), 27.45(CH₂), 26.25(CH₂),

20.24(CH₃), 20.17(CH₃), 15.07(CH₃), 11.63(indole-CH₃),

10.29(CH₃), 10.21(CH₃); HRMS (ESI): calcd for C₂₉H₃₉

N₃O₃, [M]⁺m/z, 477.2991; found, 500.2862([M+Na]⁺).

2.1.5.13.

N-Benzyl-5-[2-(diethylamino)-2-oxoethoxy]-1-

ethyl-2-methyl-1H-indole-3-carboxamide (6m)

White solid; yield: 45.9%; M.p.: 105-106°C; IR: (KBr,

cm^-1): υ 3434.3, 2925.2, 1661.7, 1617.5, 1530.2, 1478.3,

1347.7, 1259.2, 1189.2, 1139.2, 1082.9, 835.4, 756.0, 704.4;

ESI-MS, m/z: calcd. 421.24(M⁺); found 422.3([M+H]⁺),

444.3([M+Na]⁺); ¹H NMR (400 MHz, CDCl₃): δ 7.43 (d, J =

2.3 Hz, 1H, indole-H₇), 7.36 – 7.27 (m, 5H, Ar–H), 7.21 (d, J

= 8.9 Hz, 1H, indole-H₄), 6.90 (dd, J = 8.9, 2.4 Hz, 1H, in-

dole-H₆), 4.71 (d, J = 5.7 Hz, 2H, NHCH₂), 4.65 (s, 2H,

76 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

2.1.5.16. N-Benzyl-1-ethyl-2-methyl-5-[2-oxo-2-(piperidin-

1-yl)ethoxy]-1H-indole-3-carboxamide (6p)

128.60(CH-Ph), 127.87(C₉), 127.58(CH-Ph), 126.62(CH-

Ph), 126.13(CH-Ph), 122.51(CH-Ph), 121.17(CH-Ph), 120.

49(CH-Ph), 112.22(C₆), 111.11(C₃), 110.48(C₇), 103.94(C₄),

69.03(OCH₂), 68.87(C_1’’), 55.93(OCH₃), 43.67(C_2’), 38.63

(NCH₂), 38.08(NCH₂), 15.10(CH₃), 11.63(indole-CH₃);

HRMS (ESI): calcd for C₃₀H₃₃N₃O₅, [M]⁺m/z, 515.2420;

found, 516.2483([M+H]⁺), 538.2296([M+Na]⁺).

White solid; yield: 57.4%; M.p.: 89-90°C; IR: (KBr, cm^-1):

υ 3551.3, 3404.6, 3336.9, 1646.8, 1613.0, 1546.0, 1488.7,

1419.9, 1384.5, 1214.2, 1164.4, 1977.0, 984.4, 850.0, 806.9,

727.7, 695.2; ESI-MS, m/z: calcd. 433.24(M⁺); found

1

434.7([M+H]⁺); H NMR (400 MHz, CDCl₃): δ 7.43 (d, J =

2.3 Hz, 1H, indole-H₇), 7.38 – 7.25 (m, 5H, Ar–H), 7.21 (d, J

= 8.9 Hz, 1H, indole-H₄), 6.90 (dd, J = 8.8, 2.4 Hz, 1H, in-

dole-H₆), 4.71 (d, J = 5.7 Hz, 2H, NHCH₂), 4.66 (s, 2H,

OCH₂), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH₂), 3.52 (t, J =

5.3 Hz, 2H, NCH₂), 3.44 (t, J = 5.0 Hz, 2H, NCH₂), 2.71 (s,

3H, indole-CH₃), 1.66 – 1.48 (m, 6H, CH₂), 1.33 (t, J = 7.2

Hz, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 166.70(C_1’),

166.33(C_2’’), 153.68(C₅), 141.81(C₂), 139.30(C-Ph), 128.81

(C₈), 128.67(CH-Ph), 128.43(CH-Ph), 127.96(C₉), 127.41

(CH-Ph), 127.33(CH-Ph), 126.02(CH-Ph), 111.14(C₆), 110.36

(C₃), 107.65(C₇), 103.47(C₄), 68.75(C_1’’), 46.56(NCH₂),

43.69(NCH₂), 43.29(C_2’), 38.02(NCH₂), 26.62(CH₂),

25.67(CH₂), 24.60(CH₂), 15.10(CH₃), 11.63(indole-CH₃);

HRMS (ESI): calcd for C₂₆H₃₁N₃O₃, [M]⁺m/z, 433.2365;

found, 434.2433([M+H]⁺), 456.2243([M+Na]⁺).

2.1.5.19. 5-[2-(Diethylamino)-2-oxoethoxy]-1-ethyl-N-(4-

fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6s)

White solid; yield: 55.2%; M.p.: 135-137°C; IR: (KBr,

cm^-1): υ 3423.8, 3278.3, 2925.4, 1646.1, 1625.0, 1510.1,

1482.6, 1383.4, 1263.2, 1207.6, 1155.5, 1070.6, 822.5,

791.0, 706.7; ESI-MS, m/z: calcd. 439.23(M⁺); found

440.4([M+H]⁺); ¹H NMR (400 MHz, CDCl₃): δ 7.40 (dd, J =

8.5, 5.5 Hz, 2H, Ar–H), 7.29 (d, J = 2.3 Hz, 1H, indole-H₇),

7.21 (d, J = 8.9 Hz, 1H, indole-H₄), 7.02 (t, J = 8.7 Hz, 2H,

Ar–H), 6.89 (dd, J = 8.8, 2.3 Hz, 1H, indole-H₆), 4.67 (s, 2H,

OCH₂), 4.65 (d, J = 5.8 Hz, 2H, NHCH₂), 4.11 (q, J = 7.2

Hz, 2H, indole-NCH₂), 3.38 (q, J = 7.1 Hz, 4H, NCH₂), 2.71

(s, 3H, indole-CH₃), 1.32 (t, J = 7.2 Hz, 3H, CH₃), 1.17 (t, J

= 7.1 Hz, 3H, CH₃), 1.10 (t, J = 7.1 Hz, 3H, CH₃); ¹³C NMR

(101 MHz, CDCl₃): δ 167.58(C_1’), 166.31(C_2’’), 160.93(C-F),

153.79(C₅), 142.03(C₂), 135.16(C-C₆H₄F), 129.65(C₈),

129.57(CH-C₆H₄F), 129.31(CH-C₆H₄F), 125.90(C₉), 115.65

(CH-C₆H₄F), 115.43(CH-C₆H₄F), 111.14(C₆), 110.37(C₃),

107.47(C₇), 103.57(C₄), 68.72(C_1’’), 42.89(C_2’), 41.67(NCH₂),

40.38(NCH₂), 38.00(NCH₂), 15.07(CH₃), 14.43(CH₃), 12.93

(CH₃), 11.61(indole-CH₃); HRMS (ESI): calcd for C₂₅H₃₀

FN₃O₃, [M]⁺m/z, 439.2271; found, 440.2333([M+H]⁺),

462.2142([M+Na]⁺).

2.1.5.17. N-Benzyl-1-ethyl-2-methyl-5-[2-(morpholin-4-yl)-

2-oxoethoxy]-1H-indole-3-carboxamide (6q)

Yellow solid; yield: 77.7%; M.p.: 161-162°C; IR: (KBr,

cm^-1): υ 3338.5, 2969.3, 2931.8, 1663.5, 1546.4, 1481.6,

1440.8, 1279.5, 1196.4, 1161.6, 1112.1, 1035.4, 970.1,

851.3, 758.6, 707.8; ESI-MS, m/z: calcd. 435.22(M⁺); found

1

436.7([M+H]⁺); H NMR (400 MHz, CDCl₃): δ 7.44 (d, J =

2.3 Hz, 1H, indole-H₇), 7.38 – 7.27 (m, 5H, Ar–H), 7.22 (d, J

= 8.8 Hz, 1H, indole-H₄), 6.88 (dd, J = 8.8, 2.3 Hz, 1H, in-

dole-H₆), 4.71 (d, J = 5.4 Hz, 2H, NHCH₂), 4.67 (s, 2H,

OCH₂), 4.16 (q, J = 7.1 Hz, 2H, indole-NCH₂), 3.64 – 3.52

(m, 8H, CH₂), 2.72 (s, 3H, indole-CH₃), 1.34 (t, J = 7.2 Hz,

3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 167.04(C_1’),

166.24(C_2’’), 153.34(C₅), 141.80(C₂), 139.27(C-Ph), 128.85

(C₈), 128.70(CH-Ph), 127.99(CH-Ph), 127.47(C₉), 127.14

(CH-Ph), 126.97(CH-Ph), 126.11(CH-Ph), 111.05(C₆), 110.47

(C₃), 107.75(C₇), 103.43(C₄), 68.67(C_1’’), 66.95(OCH₂),

66.92(OCH₂), 46.07(NCH₂), 43.76(NCH₂), 42.52(C_2’),

38.06(NCH₂), 15.11(CH₃), 11.65(indole-CH₃); HRMS (ESI):

calcd for C₂₅H₂₉N₃O₄, [M]⁺m/z, 435.2158; found,

436.2227([M+H]⁺), 458.2037([M+Na]⁺).

2.1.5.20. 5-[2-(Dipropylamino)-2-oxoethoxy]-1-ethyl-N-(4-

fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6t)

Yellow solid; Yield: 46.9%; M.p.: 120-122°C; IR: (KBr,

cm^-1): υ 3304.4, 2963.1, 2931.9, 1658.7, 1619.4, 1510.7,

1483.0, 1423.2, 1272.9, 1207.6, 1158.4, 1091.3, 835.3,

794.0, 706.7; ESI-MS, m/z: calcd. 467.26(M⁺); found

468.4([M+H]⁺), 490.4([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.40 (dd, J = 8.5, 5.4 Hz, 2H, Ar–H), 7.29 (d, J =

2.3 Hz, 1H, indole-H₇), 7.21 (d, J = 8.9 Hz, 1H, indole-H₄),

7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H,

indole-H₆), 4.69 (s, 2H, OCH₂), 4.67 (d, J = 5.8 Hz, 2H,

NHCH₂), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH₂), 3.32 – 3.21

(m, 4H, NCH₂), 2.72 (s, 3H, indole-CH₃), 1.59 – 1.49 (m,

4H, CH₂), 1.33 (t, J = 7.2 Hz, 3H, CH₃), 0.90 (t, J = 7.4 Hz,

3H, CH₃), 0.83 (t, J = 7.4 Hz, 3H, CH₃); ¹³C NMR (101

MHz, CDCl₃): δ 168.03(C_1’), 166.33(C_2’’), 160.96(C-F),

153.81(C₅), 142.16(C₂), 135.18(C-C₆H₄F), 129.64(C₈),

129.56(CH-C₆H₄F), 129.39(CH-C₆H₄F), 125.91(C₉), 115.68

(CH-C₆H₄F), 115.47(CH-C₆H₄F), 111.03(C₆), 110.37(C₃),

107.43(C₇), 103.67(C₄), 68.71(C_1’’), 49.08(NCH₂), 47.63

(NCH₂), 42.91(C_2’), 38.03(NCH₂), 22.29(CH₂), 20.83(CH₂),

15.09(CH₃), 11.63(indole-CH₃), 11.46(CH₃), 11.38(CH₃);

HRMS (ESI): calcd for C₂₇H₃₄FN₃O₃, [M]⁺m/z, 467.2584;

found, 468.2652([M+H]⁺), 490.2463([M+Na]⁺).

2.1.5.18.

N-Benzyl-1-ethyl-5-{2-{[2-(2-methoxyphenoxy)

ethyl]amino}-2-oxoethoxy}-2-methyl-1H-indole-3-

carboxamide (6r)

White solid; yield: 86.7%; M.p.: 157-159°C; IR: (KBr,

cm^-1): υ 3455.6, 2922.3, 1686.6, 1619.2, 1508.6, 1482.9,

1384.0, 1253.7, 1209.9, 1127.8, 1027.1, 975.5, 745.5, 698.1;

ESI-MS, m/z: calcd. 515.24(M⁺); found 516.3([M+H]⁺),

538.2([M+Na]⁺); ¹H NMR (400 MHz, CDCl₃): δ 7.40 (d, J =

2.4 Hz, 1H, indole-H₇), 7.38 – 7.27 (m, 5H, benzyl-H), 7.23

(d, J = 8.8 Hz, 1H, indole-H₄), 6.89 – 6.84 (m, 5H, Ar–H),

4.68 (d, J = 5.7 Hz, 2H, NHCH₂), 4.50 (s, 2H, OCH₂), 4.14 –

4.09 (m, 4H, CH₂), 3.76 (s, 3H, OCH₃), 3.73 (t, J = 5.3 Hz,

2H, CH₂), 2.72 (s, 3H, indole-CH₃), 1.34 (t, J = 7.2 Hz, 3H,

CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 168.95(C_1’),

166.22(C_2’’), 153.06(C₅), 150.23(O-C-Ph), 147.92(O-C-Ph),

141.92(C₂), 139.14(C-Ph), 128.94(C₈), 128.60(CH-Ph),

2.1.5.21. 5-[2-(Di-sec-butylamino)-2-oxoethoxy]-1-ethyl-N-

(4-fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6u)

Yellow solid; yield: 50.7%; M.p.: 112-114°C; IR: (KBr,

cm^-1): υ 3444.7, 3295.3, 2960.1, 2921.4, 1665.1, 1631.3,

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 77

1509.6, 1483.6, 1384.2, 1213.4, 1151.4, 1090.2, 842.8,

821.8, 796.1, 749.7, 691.1; ESI-MS, m/z: calcd. 495.29(M⁺);

found 496.3([M+H]⁺), 518.3([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.40 (dd, J = 8.6, 5.4 Hz, 2H, Ar–H), 7.30 (d, J =

2.3 Hz, 1H, indole-H₇), 7.20 (d, J = 8.9 Hz, 1H, indole-H₄),

7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H,

indole-H₆), 4.73 (s, 2H, OCH₂), 4.66 (d, J = 5.9 Hz, 2H,

NHCH₂), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH₂), 3.20 – 3.15

(m, 2H, NCH), 2.71 (s, 3H, indole-CH₃), 2.08 – 1.99 (m, 4H,

CH₂), 1.19 (t, J = 7.1 Hz, 3H, CH₃), 0.93 (d, J = 6.8 Hz, 6H,

CH₃), 0.76 (t, J = 6.7 Hz, 6H, CH₃); ¹³C NMR (101 MHz,

CDCl₃): δ 168.77(C_1’), 166.32(C_2’’), 160.95(C-F), 153.73

(C₅), 142.14(C₂), 135.18(C-C₆H₄F), 129.63(C₈), 129.55(CH-

C₆H₄F), 129.03(CH-C₆H₄F), 125.91(C₉), 115.67(CH-C₆H₄F),

115.46(CH-C₆H₄F), 110.98(C₆), 110.31(C₃), 107.40(C₇),

103.86(C₄), 68.66(C_1’’), 54.43(NCH), 52.60(NCH), 42.89

(C_2’), 38.00(NCH₂), 27.42(CH₂), 26.22(CH₂), 20.22(CH₃),

20.14(CH₃), 15.06(CH₃), 11.62(indole-CH₃), 10.13(CH₃),

9.89(CH₃); HRMS (ESI): calcd for C₂₉H₃₈FN₃O₃, [M]⁺m/z,

495.2897; found, 496.2965([M+H]⁺), 518.2773([M+Na]⁺).

129.66(CH-C₆H₄F), 128.64(CH-C₆H₄F), 126.03(C₉), 115.74

(CH-C₆H₄F), 115.52(CH-C₆H₄F), 111.06(C₆), 110.54(C₃),

109.37(C₇), 103.42(C₄), 68.75(C_1’’), 66.95(OCH₂), 66.89

(OCH₂), 46.48(NCH₂), 46.08(NCH₂), 42.99(C_2’), 42.54

(NCH₂), 15.12(CH₃), 11.67(indole-CH₃); HRMS (ESI):

calcd for C₂₅H₂₈FN₃O₄, [M]⁺m/z, 453.2064; found,

454.2134([M+H]⁺), 476.1944([M+Na]⁺).

2.1.5.24. 1-Ethyl-N-(4-fluorobenzyl)-5-{2-{[2-(2-methoxy-

phenoxy)ethyl]amino}-2-oxoethoxy}-2-methyl-1H-indole-3-

carboxamide (6x)

White solid; yield: 64.9%; M.p.: 166-168°C; IR: (KBr,

cm^-1): υ 3268.1, 1673.3, 1622.3, 1507.4, 1482.0, 1254.0,

1220.4, 1125.7, 1026.3, 831.0, 739,8, 720.1; ESI-MS, m/z:

calcd. 533.23(M⁺); found 534.2([M+H]⁺), 556.2([M+Na]⁺);

¹H NMR (400 MHz, CDCl₃): δ 7.35 (dd, J = 8.6, 5.4 Hz, 2H,

4-fluorobenzyl-H), 7.23 (d, J = 2.3 Hz, 1H, indole-H₇), 7.21

(d, J = 8.9 Hz, 1H, indole-H₄), 7.03 (t, J = 8.7 Hz, 2H, 4-

fluorobenzyl-H), 6.94 (dd, J = 8.9, 2.3 Hz, 1H, NHCH₂),

6.89 – 6.82 (m, 4H, Ar–H), 4.64 (d, J = 5.8 Hz, 2H,

NHCH₂), 4.53 (s, 2H, OCH₂), 4.14 – 4.10 (m, 4H, CH₂),

3.76 (s, 3H, OCH₃), 3.74 – 3.70 (m, 2H, CH₂), 2.71 (s, 3H,

indole-CH₃), 1.34 (t, J = 7.2 Hz, 3H, CH₃); ¹³C NMR (101

MHz, CDCl₃): δ 169.02(C_1’), 166.26(C_2’’), 161.04(C-F),

153.13(C₅), 150.22(O-C-Ph), 147.86(O-C-Ph), 142.17(C₂),

135.00(C-C₆H₄F), 129.51(C₈), 129.43(CH-C₆H₄F), 128.66

(CH-C₆H₄F), 126.07(C₉), 122.58(CH-Ph), 121.19(CH-Ph),

115.81(CH-C₆H₄F), 115.71(CH-C₆H₄F), 112.21(CH-Ph),

111.11(C₆), 110.53(C₃), 110.28(CH-Ph), 107.70(C₇), 104.03

(C₄), 69.06(OCH₂), 68.99(C_1’’), 55.97(OCH₃), 42.97(C_2’),

42.81(NCH₂), 38.63(NHCH₂), 15.09(CH₃), 11.62(indole-

CH₃); HRMS (ESI): calcd for C₃₀H₃₂FN₃O₅, [M]⁺m/z,

533.2326; found, 534.2394([M+H]⁺), 556.2205([M+Na]⁺).

2.1.5.22. 1-Ethyl-N-(4-fluorobenzyl)-2-methyl-5-[2-oxo-2-

(piperidin-1-yl)ethoxy]-1H-indole-3-carboxamide (6v)

Yellow solid; yield: 42.9%; M.p.: 126-127°C; IR: (KBr,

cm^-1): υ 3513.3, 3308.9, 2918.1, 2849.7, 1655.9, 1608.0,

1544.5, 1510.5, 1439.8, 1231.5, 1210.6, 1160.6, 1087.6,

837.8, 809.2, 771.2, 689.3; ESI-MS, m/z: calcd. 451.23(M⁺);

found 452.2([M+H]⁺), 474.3([M+Na]⁺); ¹H NMR (400 MHz,

CDCl₃): δ 7.41 (dd, J = 8.4, 5.5 Hz, 2H, Ar–H), 7.28 (d, J =

2.3 Hz, 1H, indole-H₇), 7.21 (d, J = 8.8 Hz, 1H, indole-H₄),

7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.89 (dd, J = 8.8, 2.3 Hz, 1H,

indole-H₆), 4.68 (s, 2H, OCH₂), 4.66 (d, J = 5.9 Hz, 2H,

NHCH₂), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH₂), 3.52 (t, J =

5.1 Hz, 2H, NCH₂), 3.44 (t, J = 5.0 Hz, 2H, NCH₂), 2.71 (s,

3H, indole-CH₃), 1.67 – 1.52 (m, 6H, CH₂), 1.33 (t, J = 7.2

Hz, 3H, CH₃); ¹³C NMR (101 MHz, CDCl₃): δ 166.73(C_1’),

166.33(C_2’’), 161.00(C-F), 153.72(C₅), 142.08(C₂), 135.16

(C-C₆H₄F), 129.68(C₈), 129.60(CH-C₆H₄F), 129.13(CH-

C₆H₄F), 125.94(C₉), 115.67(CH-C₆H₄F), 115.46(CH-C₆H₄F),

111.12(C₆), 110.42(C₃), 107.44(C₇), 103.53(C₄), 68.82(C_1’’),

46.56(NCH₂), 46.41(NCH₂), 43.25(C_2’), 42.94(NCH₂), 26.61

(CH₂), 25.67(CH₂), 24.58(CH₂), 15.09(CH₃), 11.64(indole-

CH₃); HRMS (ESI): calcd for C₂₆H₃₀FN₃O₃, [M]⁺m/z,

451.2271; found, 452.2334([M+H]⁺), 474.2146([M+Na]⁺).

2.2. Biological Assay

For observing the antitumor activity of the target com-

pounds in vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) assay was used. A549 cells, HeLa

cells, SW480 cells, HepG2 cells and HL7702 cells were har-

vested in the logarithmic growth phase and seeded in 96-well

plates at a density of 8000 cells per well, and cultured at

37^oC in humidified atmosphere containing 5% CO₂in Dul-

becco’s modified Eagle’s medium (DMEM or RPMI-1640)

with 10% fetal bovine serum (FBS) for 24 h before any

treatments. Tested compounds were dissolved in DMSO and

diluted in the culture fluid to get various concentrations. The

cells were treated with target compounds subsequently and

incubated overnight. Then 20 μL of MTT (5 mg/mL) was

added in each well and after 4 h incubation, the medium was

removed immediately and MTT formazan was solubilized in

150 μL of DMSO. The optical densities (OD) were measured

with a microplate reader (Bio-Tek instruments, INC. USA)

at 490 nm. Inhibitory effects were determined as IC₅₀value.

2.1.5.23. 1-Ethyl-N-(4-fluorobenzyl)-2-methyl-5-[2-(morpholin-

4-yl)-2-oxoethoxy]-1H-indole-3-carboxamide (6w)

White solid; yield: 79.3%; M.p.: 151-153°C; IR: (KBr,

cm^-1): υ 3369.5, 2966.9, 2851.8, 1669.9, 1620.5, 1510.2,

1481.1, 1352.4, 1188.6, 1112.9, 1028.0, 841.8, 777.3, 707.4;

ESI-MS, m/z: calcd. 453.21(M⁺); found 454.2([M+H]⁺),

1

476.3([M+Na]⁺); H NMR (400 MHz, CDCl₃): δ 7.42 (dd, J

= 8.7, 5.4 Hz, 2H, Ar–H), 7.29 (d, J = 2.4 Hz, 1H, indole-

H₇), 7.23 (d, J = 8.9 Hz, 1H, indole-H₄), 7.04 (t, J = 8.7 Hz,

2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H, indole-H₆), 4.70 (s,

2H, OCH₂), 4.67 (d, J = 5.7 Hz, 2H, NHCH₂), 4.13 (q, J =

7.2 Hz, 2H, indole-NCH₂), 3.63 – 3.59 (m, 8H, CH₂), 2.72

(s, 3H, indole-CH₃), 1.34 (t, J = 7.2 Hz, 3H, CH₃); ¹³C NMR

(101 MHz, CDCl₃): δ 167.14(C_1’), 166.24(C_2’’), 159.20(C-F),

153.36(C₅), 142.03(C₂), 135.14(C-C₆H₄F), 129.68(C₈),

3. RESULTS AND DISCUSSION

3.1. Chemistry

As depicted in Scheme 1, a novel series of indole-3-

carboxamide derivatives were synthesized starting from the

commercially available ethyl acetoacetate (1). Reaction of

78 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

Scheme 1. Synthetic route of the target indole-3-carboxamide derivatives. Reagents and conditions: (a) R₁NH₂, rt, 3 h; (b) benzoquinone,

acetone, 30^oC, 2 h; (c) NaOH, H₂O, ethanol, reflux, 12 h; (d) EDCI, HOBt, triethylamine, DMF, CH₂Cl₂, rt, 24 h; (e) ClCH₂CONR₂R₃,

K₂CO₃, KI, DMF, 80^oC, 16 h.

ethyl acetoacetate with methylamine or ethylamine at ambi-

ent temperature afforded compound 2a or 2b, which was

converted to indole-3-carboxylate 3a or 3b via Nenitzescu

reaction with benzoquinone. Hydrolysis of compound 3a or

3b using sodium hydroxide in ethanol-aqueous solution gave

the acid 4a or 4b. Treatment of 4a or 4b with benzylamine

or 4-fluorobenzylamine in the presence of EDCI and HOBt

provided key intermediates 5a-5d. Reaction of 5a-5d with

corresponding 2-chloroacetamide derivatives by using anhy-

drous K₂CO₃and KI gave indole-3-carboxamide derivatives

6a-6x, respectively. The structures of the target compounds

were characterized by IR, ESI-MS, ¹H NMR and ¹³C NMR.

effect on the activity. However, the target compounds con-

taining [2-(2-methoxyphenoxy)ethyl]amino exhibited strong

cytotoxic activities. These results revealed that the introduc-

tion of a large aryl group was advantageous for anticancer

activity. (4) The target compounds 6c, 6i, 6j and 6u showed

excellent selective activity for HeLa cells over the other

tested cancer cells. (5) The target compound 6x (IC₅₀= 4.99,

6.81 and 13.07 μmol/L, respectively) exhibited strong cyto-

toxic activities against A549, HeLa and SW480 (6). In addi-

tion, all target compounds exhibited weak cytotoxic effects

on human liver normal cells HL7702.

3.3. Molecular Docking Study

3.2. Evaluation of the Bioactivity

Molecular docking studies were carried out to explore the

binding modes of the target compounds with the active site

of EGFR. Based on the in vitro cytotoxicity results, the tar-

get compound 6a, the moderate EGFR inhibitor and the tar-

get compound 6x, the best inhibitor in the study was selected

as the ligand example. The 3D structure of EGFR was built

as the docking model (PDB ID code: 1M17[21]).

Using erlotinib as control, the synthesized target com-

pounds 6a-6x were evaluated for the cytotoxic activities in

vitro against five cell lines (A549, HeLa, SW480, HepG2

and HL7702) by MTT colorimetric assay. The results ex-

pressed as IC₅₀values are summarized in Table 1.

The SAR analysis revealed as follows: (1) Compared to

the target compound 6f, the target compound 6r exhibited

potent cytotoxic activities against A549 and SW480 cells.

These results suggest that there was no significant difference

between methyl and ethyl in the 1-position of indole-3-

carboxamide core structure; (2) The target compound 6x

exhibited better activity than the target compound 6r against

EGFR high-expressed cancer cells. These results show that

the introduction of 4-fluorobenzylamine in 3-position was

beneficial for anticancer activity than that of benzylamine,

meanwhile also implying that the presence of fluorine atom

on benzyl group was appropriate to increase the activity. (3)

By comparison, the target compounds 6a-6f showed variable

activity. In general, the introduction of 2-(morpholin-4-yl)-2-

oxoethoxy at the 5-position resulted in weak activity. Fur-

thermore, the presence of 2-(dipropylamino)-2-oxoethoxy

and 2-(di-sec-butylamino)-2-oxoethoxy at the 5-position

resulted in a decrease or loss of the activity. In contrast, the

presence of 2-(diethylamino)-2-oxoethoxy and 2-oxo-2-

(piperidin-1-yl)ethoxy played no significant effect or slight

The binding modes of lead compound N-(3-

bromophenyl)-6-methoxy-9H-pyrimido[4,5-b]indol-4-amine

and the target compound 6a are shown in Fig. (3). As de-

picted in Fig. (3), the lead compound and the target com-

pound 6a could pretty nearly overlap in the binding model. It

is demonstrated that the docking mode of the target com-

pound 6a was similar to the lead compound by analysis of

the binding mode of the target compound 6a in the active

binding site.

The most active compound 6x was further docked into

the binding site of EGFR and the theoretical binding mode is

shown in Fig. (4). As shown in Fig. (4), the fluorine atom of

6x formed a hydrogen bond with residue Thr766 and the 5-

acetoxyl group of 6x formed a hydrogen bond with residue

Ala698. The binding modes also showed that 3-carboxamide

and indole part formed weak hydrophobic interactions with

residues Leu768, Ala719, Lys721, Val702 and Leu694. The

molecular docking study and above-mentioned results of

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 79

Table 1. In vitro cytotoxicity of the target compounds.

IC₅₀(μM)

Compd.

A549

HeLa

SW480

HepG2

HL7702

O

N

H

N

39.18

23.97

62.01

>100

N

6a

6b

6c

6d

O

N

H

>100

38.47

>100

6.34

>100

16.17

22.72

>100

78.22

47.67

>100

1.66

>100

48.29

31.38

10.57

>100

73.35

57.15

>100

86.10

67.83

>100

97.63

>100

N

O

N

H

N

O

N

H

N

O

N

H

O

N

6e

6f

O

N

H

N

H

OCH₃

N

O

N

O

N

F

H

22.69

>100

N

6g

O

N

O

N

F

H

N

6h

6i

O

N

O

N

H

>100

N

(Table 1) contd….

80 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

IC₅₀(μM)

Compd.

A549

HeLa

SW480

HepG2

HL7702

O

N

H

F

N

54.77

9.15

>100

N

6j

O

N

H

F

>100

7.40

>100

4.67

>100

27.47

>100

O

N

6k

O

N

H

F

N

H

OCH₃

N

6l

6m

6n

O

N

H

N

>100

66.75

58.02

>100

62.78

24.32

52.33

57.66

>100

N

O

N

H

N

O

N

H

N

6o

6p

6q

6r

O

N

H

>100

5.35

>100

65.74

>100

11.59

>100

83.27

>100

N

O

N

H

N

O

N

O

N

H

OCH₃

N

(Table 1) contd….

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 81

IC₅₀(μM)

Compd.

A549

HeLa

SW480

HepG2

HL7702

O

N

H

F

N

>100

64.43

>100

N

6s

O

N

F

N

H

>100

54.27

42.29

>100

17.25

9.77

>100

N

6t

O

N

H

F

N

6u

O

N

F

N

H

61.47

>100

N

6v

O

N

H

F

O

N

6w

O

N

H

F

N

H

4.99

6.81

13.07

16.72

>100

OCH₃

N

6x

（-）

Erlotinib

13.41

15.34

cancer cell lines and weak activity against the EGFR low-

expressed cancer cell lines and human liver normal cells,

which was a supporting evidence for the target compounds

as EGFR inhibitors with low toxicity. The target compounds

6c, 6f, 6i, 6j, 6l, 6r, 6u and 6x demonstrated an excellent

strong anticancer activities (6x showed equal activity to er-

lotinib). What’s more, a new EGFR inhibitor scaffold and a

preliminary discussion on their SARs provide promising

opportunities to guide further research on indole-3-

carboxamide derivatives as novel anticancer agents.

SARs analysis could contribute to the rational design of po-

tential EGFR inhibitors.

CONCLUSION

In summary, a novel series of indole-3-carboxamide de-

rivatives were designed and synthesized. All of the target

compounds were evaluated against four cancer cell lines and

one human liver normal cell lines. Some target compounds

showed moderate to strong activity as compared to the posi-

tive control erlotinib against the EGFR highly-expressed

82 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1

Zhang et al.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or

otherwise.

ACKNOWLEDGEMENTS

This work was supported by the National Science Foun-

dation of China (NFSC) for the grant No. 21342006. The

authors would like to thank Molegro ApS for kindly provid-

ing a free evaluation copy of their software package.

REFERENCES

Fig. (3). Binding sites of the target compound 6a with EGFR. The

binding modes of lead compound and 6a were denoted by yellow

and green, respectively. (The color version of the figure is available

in the electronic copy of the article).

[1]

[2]

[3]

Debnath, S.; Debnath, T.; Majumdar, S.; Arunasree, M.K.; Aparna,

V. A combined pharmacophore modeling, 3D QSAR, virtual

screening, molecular docking, and ADME studies to identify poten-

tial HDAC8 inhibitors. Med. Chem. Res., 2016, 25, 2434-2450.

Wu, S.G.; Chang, Y.L.; Yu, C.J.; Yang, P.C.; Shih, J.Y. The Role

of PIK3CA mutations among lung adenocarcinoma patients with

primary and acquired resistance to EGFR tyrosine kinase inhibi-

tion. Sci. Rep., 2016, 6, 1-9.

Mok, T.S.; Wu, Y.L.; Thongprasert, S.; Yang, C.H.; Chu, D.T.;

Saijo, N.; Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y.;

Nishiwaki, Y.; Ohe, Y.; Yang, J.J.; Chewaskulyong, B.; Jiang, H.;

Duffield, E.L.; Watkins, C.L.; Armour, A.A.; Fukuoka, M. Gefit-

inib or carboplatin-paclitaxel in pulmonary adenocarcinoma. New

Engl. J. Med., 2009, 361, 947-957.

Lynch, T.J.; Bell, D.W.; Sordella, R.; Gurubhagavatula, S.; Oki-

moto, R.A.; Brannigan, B.W.; Harris, P.L.; Haserlat, S.M.; Supko,

J.G.; Haluska, F.G.; Louis, D.N.; Christiani, D.C.; Settleman, J.;

Haber, D.A. Activating mutations in the epidermal growth factor

receptor underlying responsiveness of non-small-cell lung cancer to

gefitinib. New. Engl. J. Med., 2004, 350, 2129-2139.

Sheng, Q.; Liu, J. The therapeutic potential of targeting the EGFR

family in epithelial ovarian cancer. Brit. J. Cancer, 2011, 104,

1241-1245.

[4]

[5]

[6]

Wieduwilt, M.J.; Moasser, M.M. The epidermal growth factor

receptor family: Biology driving targeted therapeutics. Cell Mol.

Life Sci., 2008, 65, 1566-1584.

[7]

[8]

Koutras1, A.K.; Evans, T.R.J. The epidermal growth factor recep-

tor family in breast cancer. Onco. Targets Ther., 2009, 1, 5-19.

Mozzi, A.; Forcella, M.; Riva, A.; Difrancesco, C.; Molinari, F.;

Martin V.; Papini, N.; Bernasconi B.; Nonnis, S.; Tedeschi, G.;

Mazzucchelli, L.; Monti, E.; Fusi, P.; Frattini, M. NEU3 activity

enhances EGFR activation without affecting EGFR expression and

acts on its sialylation levels. Glycobiology, 2015, 25, 855-868.

Hasegawa, Y.; Takahashi, M.; Ariki, S.; Asakawa, D.; Tajiri, M.;

Wada, Y.; Yamaguchi, Y.; Nishitani, C.; Takamiya, R.; Saito, A.;

Uehara, Y.; Hashimoto, J.; Kurimura, Y.; Takahashi, H.; Kuroki,

Y. Surfactant protein D suppresses lung cancer progression by

downregulation of epidermal growth factor signaling. Oncogene,

2015, 34, 838-845.

[9]

[10]

Costa, D.B.; Kobayashi, S.S. Whacking a molecule: Clinical activ-

ity and mechanisms of resistance to third generation EGFR inhibi-

tors in EGFR mutated lung cancers with EGFR-T790M. Transl.

Lung Cancer Res., 2015, 4, 809-815.

Pao, W.; Chmielecki, J. Rational, biologically based treatment of

EGFR-mutant non-small-cell lung cancer. Nat. Rev. Cancer, 2010,

10, 760-774.

Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria,

I.; Singh, B.; Heelan, R.; Rusch V., Fulton, L.; Mardis, E.; Kupfer

D.; Wilson, R.; Kris, M.; Varmus, H. EGF receptor gene mutations

are common in lung cancers from ‘‘never smokers’’ and are associ-

ated with sensitivity of tumors to gefitinib and erlotinib. P. Natl.

Acad. Sci. USA., 2004, 101, 13306-13311.

[11]

[12]

Fig. (4). (a) Location of the target compound 6x at the interface of

EGFR subunit. (b) The binding mode of 6x in the active site of

EGFR. H-bonding interactions between 6x and EGFR were denoted

by dashed lines in green. (The color version of the figure is avail-

able in the electronic copy of the article).

[13]

[14]

Paez, J.G.; Jänne, P.A.; Lee, J.C.; Tracy, S.; Greulich, H.; Gabriel,

S.; Herman, P.; Kaye, F.J.; Lindeman, N.; Boggon, T.J.; Naoici, K.;

Sasaiti, H.; Fujii, Y.; Eck, M.J.; Sellers, W.R.; Johnson, B.E.; Mey-

erson, M. ECFR mutations in lung cancer: Correlation with clinical

response to gefitinib therapy. Science, 2004, 304, 1497-1500.

Lynch, T.J.; Bell, D.W.; Sordella, R.; Gurubhagavatula, S.; Oki-

moto, R.A.; Brannigan, B.W.; Harris, P.L.; Haserlat, S.M.; Supko,

CONSENT FOR PUBLICATION

Not applicable.

Design, Synthesis and Biological Evaluation of a Novel Series

Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 83

J.G.; Haluska, F.G.; Louis, D.N.; Christiani, D.C.; Settleman, J.;

Haber, D.A. Activating mutations in the epidermal growth factor

receptor underlying responsiveness of non-small-cell lung cancer to

gefitinib. N. Engl. J. Med., 2004, 350, 2129-2139.

cer cells by targeted delivery of 5-FU with EGF grafted hollow

mesoporous silica nanoparticles. Nanoscale, 2015, 7, 14080-14092.

Lin, W.J.; Kao, L.T. Cytotoxic enhancement of hexapeptide-

conjugated micelles in EGFR high-expressed cancer cells. Expert

Opin. Drug Del., 2014, 11, 1537-1550.

Velezheva, V.S.; Kornienko, A.G.; Topilin, S.V.; Turashev, A.D.;

Peregudov, A.S.; Brennan, P.J. Lewis acid catalyzed nenitzescu in-

dole synthesis. J. Heterocyclic Chem., 2006, 43, 873-879.

Patrick, J.B.; Saunders. E.K. Studies on the nenittzescu synthesis of

5-hydroxyindoles. Tetrahedron Lett., 1979, 20, 4009-4012.

Stamos, J.; Sliwkowski, M.X.; Eigenbrot, C. Structure of the epi-

dermal growth factor receptor kinase domain alone and in complex

with a 4-anilinoquinazoline inhibitor. J. Biol. Chem., 2002, 277,

46265-46272.

[18]

[19]

[15]

Showalter, H.D.H.; Bridges, A.J.; Zhou, H.; Sercel, A.D.;

McMichael, A.; Fry, D.W. Tyrosine kinase inhibitors. 16. 6,5,6-

tricyclic benzothieno[3,2-d]pyrimidines and pyrimido[5,4-b]- and -

[4,5-b]indoles as potent inhibitors of the epidermal growth factor

receptor tyrosine kinase. J. Med. Chem., 1999, 42, 5464-5474.

Zhou, X.F.; Guo, J.L.; Ji, Y.M.; Zhao, J.P. Reciprocal negative

regulation between EGFR and DEPTOR plays an important role in

the progression of lung adenocarcinoma. Mol. Cancer Res., 2016,

14, 448-457.

[20]

[21]

[16]

[17]

Chen, L.J.; She, X.D.; Wang, T.; He, L.; Shigdar, S.; Duan, W.;

Kong, L.X. Overcoming acquired drug resistance in colorectal can-

Article Doi

Design, synthesis and biological evaluation of a novel series of indole-3-carboxamide derivatives for cancer treatment as EGFR inhibitors

DOI: 10.2174/1570180814666170929093258

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